Sperm cryoprotective media

ABSTRACT

Described herein is a composition that comprises a cryoprotectant; a membrane protectant that stabilizes or assists in stabilization of membranes of sperm; and a free radical scavenger (e.g., a reducing agent, an antioxidant).

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/811,968, filed Jun. 12, 2007, which claims priority to U.S.Provisional Application Nos. 60/812,833, filed Jun. 12, 2006;60/840,744, filed Aug. 29, 2006; and 60/854,501, filed Oct. 25, 2006.The entire content of all of which is incorporated herein by referencein their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.RR01262-24, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Revolutionary advances in genome research and the ability to creategenetically specific strains of mice have resulted in an exponentialincrease in the number of mouse strains available for biomedicalresearch. These newly created strains, particularly those in which thestrain or mutation can be maintained by haploid germplasm, are mostefficiently preserved by freezing and storing sperm. The methodscurrently employed for cryopreservation of mouse sperm display variablymoderate success, are dependent on the genetic constitution of the maleand have, thus, in general, been deemed inadequate for the safepreservation of most inbred mouse strains. Additional methods forcryopreservation of sperm, which are less adversely affected by geneticbackground, are needed.

SUMMARY OF THE INVENTION

The cryoprotective media and methods of the present invention, whichinclude methods of cryogenically preserving sperm (e.g., mammalian,including human and non human mammals, such as rodent, including mouseand rat, sperm) are of great interest because they consistently increasethe success rates obtained with cryopreserved sperm. As used herein, theterm sperm refers to sperm and spermatozoa. The cryopreservation media(also referred to as cryoprotective media) of the invention comprises,in one embodiment, a cryoprotectant (also referred to as acryoprotective agent); a membrane protectant that stabilizes or assistsin stabilization of membranes of sperm; and a free radical scavenger(e.g., a reducing agent, an antioxidant). This cryoprotection media isreferred to herein as CPM.

In an alternative embodiment, the cryopreservation media includes two ofthe above-listed components: a cryoprotectant (CP) and a free radicalscavenger (FRS). This cryopreservation media is referred to as CP-FRS.

In a further embodiment, the cryoprotection media includes a freeradical scavenger. This cryoprotection media is referred to as FRS.

As used herein, the term “a” includes or refers to “at least one” andalso “one or more.” In all embodiments, additional substances (e.g., asolvent or liquid, such as water or a physiological buffer;anti-bacterial agent(s); antibiotic(s); antiviral agent(s)) can beincluded, as needed.

In particular embodiments, such as those in which rodent (e.g., mouse,rat), bovine or human sperm are cryopreserved, the cryopreservationmedia (CPM) used comprises a (at least one; one or more) cryoprotectant,such as a sugar; a (at least one; one or more) membrane protectant; anda free radical scavenger (at least one; one or more), such as a reducingagent, such as monothioglycerol (MTG) and/or an antioxidant, such asreduced glutathione (GSH). In specific embodiments, the sugar is atrisaccharide, such as raffinose, or a disaccharide, such as lactose. Inadditional embodiments, the cryoprotectant is glycerol or propyleneglycol, which can be used alone or in combination with each other(glycerol/propylene glycol together) or in combination with raffinoseand/or lactose (e.g., raffinose and/or lactose in combination withglycerol and/or propylene glycol). In specific embodiments, the membraneprotectant is a protein, a non-protein or a combination of a protein anda non-protein. In specific embodiments, the membrane protectant is milk,skim milk, egg yolk or a component of/in/derived from any of theforegoing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that monothioglycerol enhances the fertility ofcryopreserved C57BL/6J sperm;

FIG. 2 illustrates that GSH enhances the fertility of cryopreservedC57BL/6J sperm;

FIG. 3 illustrates that MTG reduces the ROS in cryopreserved sperm fromboth C57BL/6J (B6) and BALB/cByJ (BALB/cBy) mice;

FIG. 4 illustrates an enhancement of sperm fertilization capabilityafter cryopreservation;

FIGS. 5A-5G demonstrate that the presence of monothioglycerol (MTG)during bovine sperm cryopreservation enhances post-thaw viability. Spermfrom three collections from each of 6 different bulls were frozen in thepresence of MTG at the concentrations indicated on the x-axes. For eachcollection, two sets of three straws each were created and analyzedtwice for sperm viability. Differences in sperm cell viability among thetreatments were determined using analysis of variance on arcsinetransformed percents and preplanned comparisons to the control of no MTGusing Dunnett's method. Panel A illustrates the mean and standard errorsfor the 6 bulls while panels B though G illustrate the percent of viablesperm for the individuals. An overall treatment affect was noted as weredifferences within those bulls shown in panels B, C, and D;

FIG. 6 compares frozen versus fresh monothioglycerol and its effects onfertilization rate;

FIGS. 7A and 7B show results of assessments carried out to identifysuitable free radical scavengers; and

FIG. 8 shows results of work in which sperm from C57BL/6J were frozen in18% raffinose supplemented with 477 μM monothioglycerol. The sperm werethawed and used for in vitro fertilization using oocytes fromsuperovulated C57BL/6J females. With this approach viable sperm wereobtained and fertilized 30% of the oocytes.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is a composition, referred to as cryoprotective mediaor cryoprotection media, useful for cryopreservation of sperm from awide variety of mammals and non-mammals. In one embodiment, thecryoprotective media is referred to as CPM and comprises acryoprotectant; a membrane protectant that stabilizes or assists instabilization of membranes of sperm; and a free radical scavenger (e.g.,a reducing agent, an antioxidant). As used herein, with reference toCPM, CP-FRS and FRS (all types of cryoprotective media describedherein), the term “free radical scavenger” refers to a substance oragent that removes or inactivates free radicals, such as a substance oragent added to a mixture in order to remove or inactivate free radicals.As used herein, with reference to CPM, CP-FRS and FRS (all types ofcryoprotective media described herein), the term “reducing agent” refersto an element or a compound which, in a redox (reduction-oxidation)reaction, reduces another species (element or compound). As used herein,with reference to CPM, CP-FRS and FRS (all types of cryoprotective mediadescribed herein), the term “antioxidant” refers to a substance or agentthat reduces oxidative damage to cells and biomolecules. The compositiontypically additionally comprises water in sufficient quantity tomaintain functionality of sperm. Alternatively, a liquid or solventother than water, such as saline, or a physiological buffer, such asphosphate (PBS), bicarbonate, HEPES, MOPS, CHES, MES, CAPS or Tris, canbe used, alone or in combination with water.

In another embodiment, the cryoprotective media includes acryoprotectant and a free radical scavenger and does not include amembrane protectant. The cryoprotective media, referred to herein asCP-FRS, is useful for cryopreservation of sperm from a wide variety ofmammals and non-mammals. In a further embodiment, the cryoprotectivemedia includes a free radical scavenger and does not include acryoprotectant or a membrane protectant. In these embodiments, thecomposition typically additionally comprises water in sufficientquantity to maintain functionality of sperm, as well as additionalcomponents, as described herein.

The composition can be used for cryopreservation of sperm, includingmammalian sperm, such as rodent (e.g., mouse, rat), bovine, canine,feline, swine, goat, sheep, rabbit, horse, camelida, pet or companionmammal and human sperm, as well as non-mammalian sperm, such as avian(e.g., chicken, turkey, pet or companion bird) and fish (those producedfor consumption, pet or companion fish) sperm. The cryoprotective mediais useful for cryopreservation of sperm obtained from vertebrates of alltypes, such as mammals, including genetically modified vertebrates ofall types, including genetically modified mammals (e.g., sperm obtainedfrom genetically modified rodent (e.g., mouse, rat), bovine, canine,feline, swine, goat, sheep, rabbit, horse, camelida, other pet orcompanion mammal), as well as genetically modified non-mammals (such assperm obtained from genetically modified birds (e.g., chicken, turkey,companion bird) and fish (those produced for consumption, companionfish)). The composition described herein can be used in any method forcryogenically preserving sperm.

In one embodiment, the method of cryogenically preserving sperm(producing cryogenically preserved sperm) comprises (a) combining spermto be cryogenically preserved and a composition that comprises at leastone of the following: (1) a cryoprotectant; (2) a membrane protectantthat stabilizes or assists in stabilization of membranes of sperm; and(3) a free radical scavenger (such as a reducing agent or anantioxidant), to produce a combination (cryoprotective media-spermcombination, also referred to as the initial combination or thecombination to be cryopreserved) and (b) subjecting the combination toconditions that result in cryopreservation of sperm in the combination,thereby producing a cryogenically preserved combination that comprisescryogenically preserved sperm.

In a specific embodiment, the method of cryogenically preserving sperm(producing cryogenically preserved sperm) comprises (a) combining spermto be cryogenically preserved and a composition, referred to as CPM,that comprises: (1) a cryoprotectant; (2) a membrane protectant thatstabilizes or assists in stabilization of membranes of sperm; and (3) afree radical scavenger (such as a reducing agent or an antioxidant), toproduce a combination (cryoprotective media-sperm combination, alsoreferred to as the initial combination or the combination to becryopreserved) and (b) subjecting the combination to conditions thatresult in cryopreservation of sperm in the combination, therebyproducing a cryogenically preserved combination that comprisescryogenically preserved sperm.

The initial combination (the combination to be cryopreserved) isproduced prior to cryopreservation and can be subjected to conditionsthat result in cryopreservation of sperm in the combination immediatelyafter production or within a limited period after it is produced.Cryopreservation can be carried out at any time after production thatdoes not adversely affect the viability of the sperm, such as, for mousesperm, any time within about 90 minutes after it is produced. Theresulting sperm are cryopreserved sperm that exhibit consistently betterfertilization potential than the fertilization potential ofcryopreserved sperm obtained using presently available methods andmedia. Preservation can be carried out at a temperature of from aboutplus 4° C. to about minus 200° C. In specific embodiments,cryopreservation is carried out at a temperature between from aboutminus 80° C. to about minus 200° C. In all embodiments described herein,the resulting cryopreserved sperm can be stored indefinitely at anappropriate temperature, known to those of skill in the art (e.g., attemperatures that range from about minus 80° C. to about minus 200° C.).

The fertilization capacity or ability of sperm can be assessed usingmethods known to those of skill in the art, such as by an in vitromethod (e.g., by assessing the ability to fertilize oocytes with whichthey are combined/incubated (their ability to form 2-cell embryos, forexample)) and/or by an in viva method (e.g., by assessing the productionof offspring by females into whom the fertilized oocytes are implanted).The method can be used to cryogenically preserve sperm obtained fromvertebrates, including mammals and nonmammals, of all types, includingsperm obtained from genetically modified vertebrates (e.g., geneticallymodified mammals, such as genetically modified rodents (e.g., mouse,rat), bovine, canine, feline, swine, goat, sheep, rabbit, horse,camelida, pet or companion mammal) and genetically modified non-mammals(such as genetically modified birds (e.g., chicken, turkey, companionbird) and fish (those produced for consumption, companion fish). Referto Table 1, which is a list of animals for which the present inventioncan be used.

In certain embodiments, the method of cryogenically preserving spermcomprises: (a) combining sperm to be cryogenically preserved and acomposition that comprises (1) a cryoprotectant, such as one or moresugars (e.g., raffinose, lactose), glycerol and/or propylene glycol; (2)a membrane protectant, which is a macromolecule or mixture ofmacromolecules that stabilizes or assists in stabilization of membranesof sperm (e.g., milk or milk powder, such as skim milk or skim milkpowder; a protein in milk; a milk protein or component thereof); and (3)a free radical scavenger, such as a reducing agent or an antioxidant(e.g., monothioglycerol, reduced glutathione (GSH)), to produce acombination (cryoprotective media-sperm combination); and (h) subjectingthe combination to conditions that result in cryopreservation of spermin the combination, thereby producing a cryopreserved combination thatcomprises cryopreserved sperm. The macromolecule(s) used can be anymacromolecule described herein or any other macromolecule that has thedesired characteristics. The initial combination (the combination to becryopreserved) is produced prior to cryopreservation and can besubjected to conditions that result in cryopreservation of sperm in thecombination immediately after it is produced or within a limited periodafter it is produced. Cryopreservation can be carried out at any timeafter production that does not significantly adversely affect theviability of the sperm. For example, with mouse or rat sperm,cryopreservation can be carried out at any time within about 90 minutesafter it is produced. As a further example, cryopreservation of human orbovine sperm can be carried out up to about 24 hours after production ofthe combination. Preservation is carried out at a temperature of fromabout plus 4° C. to about minus 200° C. In specific embodiments,cryopreservation is carried out at a temperature between from aboutminus 80° C. to about minus 200° C. Before the sperm is incubated withoocytes, the sperm is thawed and may also be washed. In all embodimentsdescribed herein, the resulting cryopreserved sperm can be storedindefinitely. The resulting sperm are cryopreserved sperm that exhibitconsistently better fertilization potential than the fertilizationpotential of cryopreserved sperm obtained using presently availablemethods and media.

The fertilization capacity or ability of sperm can be assessed usingmethods known to those of skill in the art, such as in vitro, byassessing the ability to fertilize oocytes with which they arecombined/incubated (their ability to form 2-cell embryos, for example)and/or in vivo by assessing the production of offspring by females intowhom the fertilized oocytes are implanted. In the case of human sperm,fertilization capacity or ability can be assessed using availablemethods, such as a functional assay, including, but not limited to amotility assay, a viability assay, a hemizona assay (binding of thesperm to the zona pellucida) or sperm penetration into zona-free hamsteroocytes.

In a specific embodiment, the method of cryogenically preserving spermcomprises: (a) combining sperm to be cryogenically preserved and acomposition that comprises (1) a cryoprotectant, which is one or moresugars (e.g., raffinose, lactose); (2) a membrane protectant, which is amacromolecule such as a protein (e.g., milk or a protein contained inmilk; skim milk or a protein contained in skim milk, egg yolk or aprotein contained in egg yolk) that stabilizes or assists instabilization of membranes of sperm; and (3) a free radical scavenger,such as a reducing agent or an antioxidant (e.g., monothioglycerol,reduced glutathione (GSH)), to produce a combination (cryoprotectivemedia-sperm combination); and (b) subjecting the combination toconditions that result in cryopreservation of sperm in the combination,thereby producing a cryopreserved combination that comprisescryopreserved sperm. The initial combination (the combination to becryopreserved) is produced prior to cryopreservation and can besubjected to conditions that result in cryopreservation of sperm in thecombination immediately after it is produced or within a limited periodafter it is produced. Cryopreservation can be carried out at any timeafter production that does not significantly adversely affect theviability of the sperm. For example, with mouse sperm, cryopreservationcan be carried out at any time within about 90 minutes after it isproduced. Preservation is carried out, for example, at a temperature offrom about plus 4° C. to about minus 200° C. In specific embodiments,cryopreservation is carried out at a temperature between from aboutminus 80° C. to about minus 200° C. In all embodiments described herein,the resulting cryopreserved sperm can be stored indefinitely. Theresulting sperm are cryopreserved sperm that exhibit consistently betterfertilization potential than the fertilization potential ofcryopreserved sperm obtained using presently available methods andmedia. The fertilization capacity or functionality of sperm can beassessed using methods known to those of skill in the art, such as invitro, by assessing the ability to fertilize oocytes with which they arecombined/incubated (their ability to form 2-cell embryos, for example)and/or in vivo by assessing the production of offspring by females intowhom the fertilized oocytes are implanted. In the case of human sperm,fertilization capacity or ability can be assessed using availablemethods, such as a functional assay, including, but not limited to, ahemizona assay (binding of the sperm to the zona pellucida) or spermpenetration into zona-free hamster oocytes.

In an alternative embodiment, the cryoprotective media includes a (atleast one; one or more) cryoprotectant and a (at least one; one or more)free radical scavenger and does not include a membrane protectant. Thiscryoprotective media, referred to herein as CP-FRS, is useful for thesame purposes and in the methods as described herein for CPM.

In one embodiment, the method of cryogenically preserving sperm(producing cryogenically preserved sperm) comprises (a) combining spermto be cryogenically preserved and a composition that includes acryoprotectant and a free radical scavenger (such as a reducing agent oran antioxidant), to produce a combination (cryoprotective media-sperm orFRS-sperm combination, also referred to as the initial combination orthe combination to be cryopreserved) and (b) subjecting the combinationto conditions that result in cryopreservation of sperm in thecombination, thereby producing a cryogenically preserved combinationthat comprises cryogenically preserved sperm.

In a specific embodiment, the method of cryogenically preserving sperm(producing cryogenically preserved sperm) comprises (a) combining spermto be cryogenically preserved and a composition, referred to as CP-FRS,that includes a cryoprotectant and a free radical scavenger (such as areducing agent or an antioxidant), to produce a combination(cryoprotective media-sperm combination, also referred to as the initialcombination or the combination to be cryopreserved) and (b) subjectingthe combination to conditions that result in cryopreservation of spermin the combination, thereby producing a cryogenically preservedcombination that comprises cryogenically preserved sperm.

The initial combination (the combination to be cryopreserved) isproduced prior to cryopreservation and can be subjected to conditionsthat result in cryopreservation of sperm in the combination immediatelyafter production or within a limited period after it is produced.Cryopreservation can be carried out at any time after production thatdoes not adversely affect the viability of the sperm, such as, for mousesperm, any time within about 90 minutes after it is produced. Theresulting sperm are cryopreserved sperm that exhibit consistently betterfertilization potential than the fertilization potential ofcryopreserved sperm obtained using presently available methods andmedia. Preservation can be carried out at a temperature of from aboutplus 4° C. to about minus 200° C. In specific embodiments,cryopreservation is carried out at a temperature between from aboutminus 80° C. to about minus 200° C. In all embodiments described herein,the resulting cryopreserved sperm can be stored indefinitely at anappropriate temperature, known to those of skill in the art (e.g., attemperatures that range from about minus 80° C. to about minus 200° C.).

The cryoprotectants and free radical scavengers described herein areuseful in the CP-FRS composition and methods in which CP-FRS is used. Ina further embodiment, the cryopreservation media includes a (at leastone, one or more) free radical scavenger and does not include acryoprotectant or a membrane protectant. In one embodiment, the methodof cryogenically preserving sperm (producing cryogenically preservedsperm) comprises (a) combining sperm to be cryogenically preserved and acomposition that includes a free radical scavenger (such as a reducingagent or an antioxidant), to produce a combination (cryoprotectivemedia-sperm or FRS-sperm combination, also referred to as the initialcombination or the combination to be cryopreserved) and (b) subjectingthe combination to conditions that result in cryopreservation of spermin the combination, thereby producing a cryogenically preservedcombination that comprises cryogenically preserved sperm.

In a specific embodiment, the method of cryogenically preserving sperm(producing cryogenically preserved sperm) comprises (a) combining spermto be cryogenically preserved and a composition, referred to as FRS,that includes a free radical scavenger (such as a reducing agent or anantioxidant), to produce a combination (cryoprotective media-spermcombination, also referred to as the initial combination or thecombination to be cryopreserved) and (b) subjecting the combination toconditions that result in cryopreservation of sperm in the combination,thereby producing a cryogenically preserved combination that comprisescryogenically preserved sperm. The initial combination (the combinationto be cryopreserved) is produced prior to cryopreservation and can besubjected to conditions that result in cryopreservation of sperm in thecombination immediately after production or within a limited periodafter it is produced. Cryopreservation can be carried out at any timeafter production that does not adversely affect the viability of thesperm, such as, for mouse sperm, any time within about 90 minutes afterit is produced. The resulting sperm are cryopreserved sperm that exhibitconsistently better fertilization potential than the fertilizationpotential of cryopreserved sperm obtained using presently availablemethods and media. Preservation can be carried out at a temperature offrom about plus 4° C. to about minus 200° C. In specific embodiments,cryopreservation is carried out at a temperature between from aboutminus 80° C. to about minus 200° C. The free radical scavengersdescribed herein are useful in the FRS composition and methods in whichFRS is used.

In all embodiments described herein, the resulting cryopreserved spermcan be stored indefinitely at an appropriate temperature, known to thoseof skill in the art (e.g., at temperatures that range from about minus80° C. to about minus 200° C.). In all embodiments, the cryoprotectivemedia can comprise additional components, such as water or othersolvent, anti-bacterial agent(s), anti-viral agent(s), and/orantibiotic(s).

As used herein, “a” includes “at least one” and “one or more.”

In certain embodiments, the method of the present invention is a methodof producing live offspring, which can be vertebrates of many types,including mammals and non-mammals. It is a method of producing liveoffspring, such as rodents (e.g., mouse, rat), cattle/bovine (cows,bulls), dogs, cats, pigs/swine, goats, sheep, horses, camels, rabbits,pet or companion mammals, birds (e.g., chicken, turkey, pet or companionfish (those produced for consumption, companion fish), other species(including endangered species) and humans. The method of producing liveoffspring comprises the steps described above for cryogenicallypreserving sperm and further comprises thawing the resultingcryopreserved sperm (thereby producing thawed sperm); introducing thawedsperm or fertilized oocytes produced by using thawed sperm resultingfrom the method described above into an appropriate female (e.g., apseudopregnant female, such as a pseudopregnant mouse, when fertilizedoocytes are used and a receptive female who is in oestrus, when thawedsperm are used) and maintaining the female into whom oocytes or spermwere introduced under conditions appropriate for growth and developmentof fertilized oocytes into live offspring (appropriate for production oflive offspring), whereby live offspring is/are produced. In thoseembodiments in which thawed sperm are introduced into an appropriatefemale, they are present in an appropriate media, such as the CPM or IVFmedia. For example, the thawed sperm/cryoprotective media combination isundiluted or diluted into an appropriate IVF media (e.g., Cooks MouseVitro Fert (Cook Australia; Queensland, Australia; (Quinn et al., 1995))and introduced into a suitable receptive female. The sperm can beintroduced by surgery-assisted artificial insemination (AI). Forexample, the sperm suspension is transferred by means of a transferpipette directly into a space between the ovary and ovarian bursa nearthe infundibulum of an oviduct. Alternatively, the sperm can be insertedinto the ampulla region through the oviductal wall of an appropriatereceptive female, such as a superovulated female, see (Sato and Kimura,2001; Sato et al., 2004). Alternatively, the thawed sperm/cryoprotectivemedia combination is diluted into an appropriate IVF media (e.g. CooksMouse Vitro Fert (Cook Australia; Queensland, Australia; (Quinn et al.,1995)) and is subsequently mixed with egg masses or oocytes and culturedin vitro to the fertilized egg, 2-cell stage or 4-cell to 16-cell stageembryos, morula or blastocyst stage embryos. Subsequently, fertilizedeggs, 2-cell stage or 4-cell to 16-cell stage embryos, morula orblastocyst stage embryos are introduced into an appropriate receptivefemale.

Also the subject of this invention are cryoprotective media-spermcombinations; cryogenically preserved sperm; and fertilized oocytes,2-cell embryos and zygotes produced by the methods described.

Also the subject of this invention are non-human live offspring producedby the methods described. In specific embodiments, the live offspringare rodents (e.g., mouse, rat), cows, dogs, cats, pigs/swine, goats,sheep, horses, camels, rabbits, other companion mammals, birds (e.g.,chicken, turkey, companion birds), fish (those produced for consumption,companion fish), and other species (including endangered species).

Components of the Cryoprotective Media Cryoprotectant

The cryoprotective media can be varied in its specific components. Forexample, the cryoprotectant (also referred to as a cryoprotective agent)is typically a non-penetrating cryoprotectant (at least one, one or morenon-penetrating cryoprotectant(s)). A penetrating cryoprotectant(s) (atleast one, one or more penetrating cryoprotectant(s)) can also be used.A combination of one or more non-penetrating cryoprotectant and one ormore penetrating cryoprotectant can be used.

A non-penetrating cryoprotectant is a solute (agent/compound) that isincapable of moving across a cell membrane (here, the membrane of thesperm) to any significant/substantial extent. As a component of the cellsuspension, it alters the nature and/or extent of changes that occurwithin sperm and in the cryoprotective medium to enhance or increase thenumber (percent) of cells (sperm) that survive cryopreservation.Non-penetrating cryoprotectants useful in the present invention can be asugar or a nonsugar or a combination of one or more sugars and one ormore nonsugars. In specific embodiments, this component of thecryoprotective media is a sugar or combination of two or more sugars.Sugars included in the cryoprotective media can be a disaccharide(s)(trehalose, melibiose, sucrose, lactose), a trisaccharide(s) (e.g.,raffinose, melezitose), a tetrasaccharide(s) (e.g. stachyose), anoligosaccharide(s) (mannotriose), or a polysaccharide(s) (e.g., dextran,hydroxyl-ethyl starch (HES)) and sugar alcohols thereof (e.g., maltitol,lactitol). In particular embodiments, the sugar included in thecryoprotective media is raffinose or lactose. Nonsugars, such aspolyvinyl pyrrolidone (PVP), polyethylene oxide (PEO) orpolyethyleneglycol (PEG), can be included in the cryoprotective media.For example, high molecular weight PEG can be used, such as PEG of anyaverage molecular weight from PEG400 to PEG20000 Dalton (PEG400 toPEG20000). Alternatively, a penetrating cryoprotectant can be used. Apenetrating cryoprotectant is a solute (agent/compound) or a solventthat can move across a cell membrane, such as the membrane of the sperm.This component alters the nature and/or extent of changes that occurboth within the sperm and in the cryoprotective media to enhance orincrease the percent (number) of cells (sperm) that survivecryopreservation.

In specific embodiments, this component of the cryoprotective media isone or more of the following: a penetrating sugar alcohol, such asglycerol; dimethyl sulfoxide (DMSO); an alcohol, such as ethylene glycolor propylene glycol, or a combination of glycerol and propylene glycol.A combination of one or more non-penetrating cryoprotectants and one ormore penetrating cryoprotectants can also be used. It should beunderstood that some components, such as glycerol, act as penetrating ornon-penetrating cryoprotectant, depending on the conditions under whichthey are used. Glycerol is a sugar alcohol that is penetrating at roomto body temperature and non-penetrating at temperatures below 0° C. Atambient temperatures, glycerol is a penetrating cryoprotectant.

Membrane Protectant

The membrane protectant component of the cryoprotective media can be anyof a wide variety of macromolecules that act as a buffer and diluent tostabilize or assist in stabilizing cell membranes. The membraneprotectant component can be a (one or more, at least one) protein, a(one or more, at least one) non-protein or a combination of a protein orproteins and a non-protein or non-proteins. The one or more proteins canbe present in/derived from animal sources (animal proteins) and can be,for example, milk, milk derivatives or components, such as milk protein,skim milk (such as liquid skim milk or skim milk powder), a protein inmilk (e.g., in whole milk, skim milk) or a component of a milk protein;casein, egg, egg yolk, egg white (e.g. fresh egg, egg yolk or egg white,such as fresh chicken egg, egg yolk or egg white), a protein in egg, eggyolk or egg white; collagen, elastin, gelatin, atelocollagen,fibronectin, peptones, keratin, albumin or any combination thereof.Alternatively, the one or more proteins can be derived from plantsources (plant proteins) and can be, for example, soy protein, wheatprotein, corn protein, coconut milk, Aloe vera extract, jojoba extract,or any combination thereof. The membrane protectant can be a combinationof one or more animal proteins and one or more plant proteins. The oneor more proteins can be a recombinantly produced protein, such ascasein, collagen, gelatin, fibronectin, albumin, lactalbumin,lactoglobulin, keratin or any combination thereof. Alternatively, themembrane protectant can be a lipid (e.g., animal lipid, plant lipid,chemically synthesized), synthetic lipids, such as phosphatidylglycerol,phosphatidic acid, 1,1′,2,2′-tetra-acyl-cardiolipin,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,polyoxyethylene based lipids, arachidonic linoleic, linolenic, myristic,oleic, palmitic or stearic fatty acids, cholesterol, Pluronic F-68 orany combination thereof can be used (e.g., as in chemically definedlipid concentrate supplies by Invitrogen Cat. No. 11905-031 or lipidmixture 1 by Sigma, Cat. No. L0288). A further example of a membraneprotectant is a polymer, polyvinylalcohol (PVA). Any combination of themembrane protectants described herein or similar agents can be used inthe cryoprotective media. For example, one or more proteins, such as oneor more proteins of animal origin and/or one or more proteins of plantorigin and/or one or more recombinant protein can be included in thecryoprotective media, which can also comprise additional membraneprotectants, such as one or more lipids (e.g., one or more lipids ofanimal origin and/or one or more lipids of plant origin and/or one ormore synthetic lipid, any mixture of these types of lipids) and/or PVA.The concentration of the membrane protectant can be determinedempirically, using methods known to those of skill in the art. Incertain embodiments, the concentration of membrane protectant is fromabout 1% weight/volume to about 50% weight/volume, such as 3%weight/volume for skim milk powder. Alternatively, the concentration canbe from about 1% volume/volume to about 90% volume/volume, such as 23%volume/volume for egg yolk and 90% for milk. In all embodiments, theconcentration can be any concentration within these ranges.

Free Radical Scavenger

The free radical scavenger component (also referred to as a free radicalscavenger agent) of the cryoprotective media can be any scavenger whichis compatible with viability of sperm and sufficiently effective(active) that it removes or inactivates free radicals (reactive oxygenspecies) in the combination to such an extent that the percent (number)of cells (sperm) that survive cryopreservation and can fertilize oocytesis consistently enhanced. A free radical scavenger component can be anycompound or molecule, such as a reducing agent or an antioxidant, thathas these desired characteristics. Useful free radical scavenger agentscan be identified as described in Example 4 and Example 13, in which theability of monothioglycerol to act as reducing agent was assessed usinga fluorescent dye and flow cytometry. Free radical scavenger agentsuseful in the cryoprotective media of the present invention canalternatively be identified by methods known to those of skill in theart.

One or more free radical scavengers (e.g., one or more reducing agent(s)and/or one or more antioxidants) can be included in the cryoprotectivemedia. In specific embodiments, the free radical scavenger can be, forexample, one or more reducing agents, such as one or more of thefollowing: monothioglycerol (MTG), beta-mercaptoethanol, dithiothreitol(DTT), Tris (2-carboxyethyl)phosphine (TCEP), dithioerythritol,thioredoxin (TRX), dithionite, 2-mercaptoethylamine, dimethyl thiourea;nordihydroguaiaretic acid (NDGA), 2,3-dimercapto-1-propanol orhydroquinone. Alternatively, the free radical scavenger can be one ormore antioxidants, such as one or more of the following: an amino acidor derivative thereof (e.g., reduced glutathione (GSH), cysteine,homocysteine, N-acetyl cysteine (NAC), methionine, N-2-mercaptopropionylglycine, alanine, glutathionine), bilirubin, melatonin, mannitol, lipoicacid, 10,11-dihydroxyaporphine (DHA), butylated hydroxyanisole (BHA),butylated hydroxytoluene, dihydroolipoic acid, tetrahydropapaveroline(THP), 2-thiobarbituic acid, or taurine, and dimercaptosuccinic acidinositol. Further, the free radical scavenger can be a vitamin (such asvitamin E (tocopherol or a derivative thereof, such as water solubleforms of vitamin E; vitamin C (ascorbic acid); vitamin A, carotenoids,(Astaxanthin), vitamin B complex (including inositol) or Coenzyme Q);allopurinol, dimethyl sulfoxide, deferoxamine, an enzyme (such ascatalase, glutathione peroxidase or superoxide dismutase), a steroid(such as 21-aminosteroids, methylprednisolone), or glutathionine.

In a specific embodiment of the cryoprotectant media, the free radicalscavenger is monthioglycerol. In this embodiment, the free radicalscavenger is typically present in the cryoprotective media at a level offrom about 50 μM to about 50 mM (50000 μM). Any level between from about50 μM to about 50 mM (50000 μM) monothioglycerol can be used. In afurther embodiment, the free radical scavenger is reduced glutathione(GSH). In this embodiment, the free radical scavenger is typicallypresent in the media at a level of from about 50 μM to about 50 mM. Anylevel between from about 50 μM to about 50 mM (50000 μM) GSH can beused. The concentration of other free radical scavengers can bedetermined empirically and will vary depending on the particular agentused.

In a specific embodiment of the cryoprotective media, the cryoprotectantis a sugar (e.g., raffinose or lactose), or a sugar alcohol (e.g.,glycerol), or an alcohol (e.g., propylene glycol), or combination of twoor more of the preceding (e.g. lactose and/or raffinose and propyleneglycol or lactose and/or raffinose and glycerol); the membraneprotectant is a macromolecule, such as a milk or milk protein (such asskim milk protein) or skim milk or egg yolk; and the free radicalscavenger is a reducing agent, such as monothioglycerol, or anantioxidant, such as GSH.

In an alternative embodiment of the cryoprotective media (CP-FRS), thecryoprotectant is a sugar (e.g., raffinose, or lactose), or a sugaralcohol (e.g., glycerol), or an alcohol (e.g., propylene glycol), or acombination of two or more of the preceding (e.g., lactose and/orraffinose and propylene glycol or lactose and/or raffinose and glycerol)and the free radical scavenger is a reducing agent, such asmonothioglycerol or dithiothreitol, or an antioxidant, such as GSH.

In further specific embodiments, the (at least one; one or more) sugaris raffinose or lactose; the (at least one; one or more) membraneprotectant is a macromolecule, such as a milk; and the (at least one;one or more) reducing agent is monothioglycerol (MTG). In furtherspecific embodiments, the (at least one) sugar is raffinose; the (atleast one) macromolecule is a milk protein; and the (at least one)antioxidant is GSH. Alternatively, the (at least one; one or more)membrane protectant is skim milk.

In further specific embodiments, the cryoprotectant is a (at least one;one or more) sugar, such as lactose or a sugar alcohol, such as glycerolor an alcohol, such as propylene glycol; the (at least one; one or more)membrane protectant is egg yolk (e.g., fresh egg yolk, such as freshchicken egg yolk) or skim milk; and the (at least one; one or more) freeradical scavenger is monothioglycerol (MTG) or reduced glutathione ordithiothreitol (DTT). In further specific embodiments, the (at leastone; one or more) sugar is raffinose; the (at least one; one or more)macromolecule is skim milk or a milk protein; and the (at least one; oneor more) free radical scavenger is GSH, MTG or DTT.

In a specific embodiment, the method of the present invention is amethod of cryogenically preserving sperm, comprising combining spermwith cryoprotective media which comprises at least one sugar; at leastone membrane protectant (e.g., a macromolecule such as a protein or apolymer); and at least one free radical scavenger, such as a reducingagent or an antioxidant. The at least one sugar can be any of the sugarsdescribed herein; the membrane protectant is a polymer, such aspolyvinylalcohol or Pluronic F-68; and the at least one free radicalscavenger agent can be any reducing agent or any antioxidant describedherein or a combination of one or more reducing agents and one or moreantioxidants.

The quantities and relative concentrations of each of these componentsof the cryoprotective media (cryoprotectant, membrane protectant, freeradical scavenger), as well as the quantities and relativeconcentrations of other media components (e.g., water) can be determinedempirically, using methods known to those of skill in the art, forproduction of a variety of media. See the examples.

Methods of Preserving Sperm

The cryopreservation media and methods of the present invention can beused for preserving sperm from a wide variety of mammals and in specificembodiments, is used for cryopreservation of rodent sperm, such as mouseor rat sperm; bovine sperm or human sperm. As discussed in greaterdetail below, the media and methods can be used with anystrain/substrain or stock of mouse. In specific instances, thecomposition of the cryopreservation media may be adjusted as to itscomponents and/or their respective concentrations, in order to enhancethe effectiveness of the media in protecting the particular type ofsperm (e.g., adjusted to suit the particular strain of mouse sperm) fromcryopreservation. Adjustments can be made empirically, using methodsknown to those of skill in the art. For example, the mouse strain can beeither inbred (e.g., C57BL/6; BALB/c; FVB/N; 129S3/Svlm) or outbredstock.

The cryoprotective media and methods of this invention are particularlyuseful for cryopreservation of mouse sperm, such as sperm from inbredmouse strains, particularly the C57BL mouse strains, such as C57BL/6,and, thus, for production of live mice of all types and strains, such aslive C57BL offspring, such as live C57BL/6 offspring. The method can beused to produce live offspring from all mice with C57BL background, suchas C57BL/6 strains and particularly C57BL16 offspring (e.g., geneticallymodified transgenics, knockouts, as well as, but not limited to, live129 offspring, live FVB offspring and live BALB/c offspring. See, forexample, Tables 3A and 3B).

Sperm which are processed by the method and using the cryoprotectivemedia of the present invention can be non ejaculate, such as thatobtained as described herein, or ejaculate sperm. The cryoprotectivemedia-sperm combination can be placed into vials, cryotubes, glassampoules, ministraws or straws for cryopreservation and subsequentstorage. The quantity or concentration of sperm to be used can bedetermined empirically, using known methods, taking into considerationsuch factors as the type of sperm and the type, size and shape of thecontainer being used. In the instances in which mouse sperm is beingcryopreserved or used, 0.3× to 3× of sperm per 10 microliters of CPM orCP-FRS are generally used. The concentration 1× is defined such that thesperm from one male are collected into 1 mL of CPM or CP-FRS. In thecase of C57BL/6 mice, this is a concentration in the range of31.5±2.8×10⁶ sperm/mL. For other strains and species, the optimalconcentration may be different from that for C57BL/6 sperm and can beempirically determined using techniques known to the person skilled inthe art.

Kits

In another aspect, the present invention provides any of thecompositions described herein in kits, optionally including instructionsfor use of the compositions (e.g., for preserving sperm and/or othercells). That is, the kit can include a description of use of acomposition in any method described herein. A “kit,” as used herein,typically defines a package, assembly, or container (such as aninsulated container) including one or more of the components of theinvention, and/or other components associated with the invention, forexample, as previously described. Each of the components of the kit maybe provided in liquid form (e.g., in solution), or in solid form (e.g.,a dried powder, frozen, etc.).

In some cases, the kit includes one or more components, which may bewithin the same or in two or more receptacles, and/or in any combinationthereof. The receptacle is able to contain a liquid, and non-limitingexamples include bottles, vials, jars, tubes, flasks, beakers, or thelike. In some cases, the receptacle is spill-proof (when closed, liquidcannot exit the receptacle, regardless of orientation of thereceptacle).

In some embodiments, a kit may comprise two or more of: a membraneprotectant, a free radical scavenger, and a cryoprotective agent, whichmay be in the same receptacle, or divided among two or more receptacles.As a specific, non-limiting example, a first receptacle may contain atleast two of: the membrane protectant, the free radical scavenger, andthe cryoprotective agent, while a second receptacle may contain acomponent that is not present in the first vessel. In some cases, thecomponents of the kit may be contained within a suitable container, suchas a cardboard box, a Styrofoam box, etc. The kit may be shipped at roomtemperature (about 25° C.), chilled (e.g., at about 4° C.), and/or anyone or more of the components may be shipped frozen (e.g., between −20°C. and −80° C., at about −150° C., etc.) or in liquid nitrogen (about−196° C.). In some cases, one or more of the components are frozenand/or shipped on dry ice (about −80° C.).

With regard to frozen solutions, if more than one component is present(e.g., as described above), the components may be frozen together in onecommon liquid (e.g., within one common receptacle), or as two or moreseparate liquids (e.g., within separate receptacles).

In certain cases, some of the components may be processable (e.g., to anactive form), for example, by the addition of a suitable solvent orother species, which may or may not be provided with the kit. Forexample, the component may be heated or a liquid may be added to thecomponent (e.g., if the component is frozen, lyophilized, shipped in aconcentrated form, etc.).

In one embodiment, the kit for use in cryopreserving sperm comprises atleast one cryoprotectant; at least one free radical scavenger; and atleast one membrane protectant. In a specific embodiment, the freeradical scavenger was identified by a method described herein. In otherembodiments, the kit comprises at least one cryoprotectant and at leastone free radical scavenger.

In some cases, the kit will include a cryogenic vessel which is a vesselsuitable for containing materials at cryogenic temperatures, forexample, liquid nitrogen. Those of ordinary skill in the art will beaware of suitable cryogenic vessels, for example, a Dewar flask (e.g.,formed from stainless steel and/or aluminum, etc.), a vapor shipper, astainless steel container, a Styrofoam container, or the like.Typically, cryogenic temperatures include temperatures below about −150°C., below about −170° C., or below about −190° C. For instance, liquidnitrogen has a boiling point of about −196° C.

The kit may also contain a receptacle for holding sperm and/or othercells. In some cases, this receptacle is able to contain the liquidcontaining the sperm, and/or a frozen solution or liquid containing thesperm. For example, the receptacle may be constructed so that it canwithstand cryogenic temperatures without rupture or fracture. In someembodiments, the receptacle can be placed within a cryogenic vessel, asdescribed above, (e.g., using a float (for example, that can float onliquid nitrogen or other cryogenic liquid within the cryogenic vessel)).Non-limiting examples of receptacles for sperm and/or other suitablecells include cell straws, glass ampoules, cryotubes, cryovials, etc.The receptacle may be pre-labeled in certain instances.

Examples of other compositions or components associated with theinvention include, but are not limited to, diluents, salts, buffers,chelating agents, preservatives, drying agents, antimicrobials, needles,syringes, packaging materials, tubes, bottles, flasks, beakers, and thelike, for example, for using, modifying, assembling, storing, packaging,preparing, mixing, diluting, and/or preserving the components for aparticular use. In embodiments where liquid forms of any of thecomponents are used, the liquid form may be concentrated or ready touse.

A kit of the invention generally will include instructions orinstructions to a website or other source in any form that are providedfor using the kit in connection with the components and/or methods ofthe invention. For instance, the instructions may include instructionsfor the use, modification, mixing, diluting, preserving, assembly,storage, packaging, and/or preparation of the components and/or othercomponents associated with the kit. In some cases, the instructions mayalso include instructions for the delivery of the components, forexample, for shipping at room temperature, sub-zero temperatures,cryogenic temperatures, etc. The instructions may be provided in anyform that is useful to the user of the kit, such as written or oral(e.g., telephonic), digital, optical, visual (e.g., videotape, DVD,etc.) and/or electronic communications (including Internet or web-basedcommunications), provided in any manner.

As used herein, instructions can include protocols, directions, guides,warnings, labels, notes, and/or “frequently asked questions” (FAQs), andtypically involve written instructions on or associated with theinvention and/or with the packaging of the invention. Instructions canalso include instructional communications in any form (e.g., oral,electronic, digital, optical, visual, etc.), provided in any manner(e.g., within or separate from a kit) such that a user will clearlyrecognize that the instructions are to be used with the kit.

As an example, a kit as discussed herein may be shipped to a user,typically with instructions for use. For instance, the instructions mayinstruct the user to add sperm to the membrane protectant and the freeradical scavenger, and store the resulting combination and/or return thekit and the sperm to the sender. As another example, the instructionsmay instruct the user to combine of sperm, membrane protectant, freeradical scavenger, and cryoprotective agent, and cryopreserve theresulting combination (e.g., as described above). The cryopreservedcombination could then be stored, returned to the shipper for storageand later recovery, or the like.

Identification of Free Radical Scavengers

One embodiment is a method of identifying a free radical scavengersuitable for use in cryopreserving sperm, comprising assessing at leastone of the following activities of a candidate free radical scavenger:(a) capability (ability) to reduce reactive oxygen species in sperm; (b)effect of sperm motility; and (c) effect on in vitro fertilization,wherein if the candidate free radical scavenger produces a beneficialeffect on at least one of (a), (b) or (c), the candidate free radicalscavenger is a free radical scavenger. For example, if there is areduction in reactive oxygen species in sperm when the sperm arecontacted, combined or incubated with a candidate free radicalscavenger, the candidate free radical scavenger is a free radicalscavenger suitable for use in cryopreserving sperm. If sperm motility isenhanced or at least maintained at substantially the same level as priorto cryopreservation, when the sperm are contacted, combined or incubatedwith a candidate free radical scavenger, the candidate free radicalscavenger is a free radical scavenger suitable for use in cryopreservingsperm. If in vitro fertilization is enhanced or at least maintained atsubstantially the same level as prior to cryopreservation, when thesperm are contacted, combined or incubated with a candidate free radicalscavenger, the candidate free radical scavenger is a free radicalscavenger suitable for use in cryopreserving sperm. In one embodiment,if a free radical scavenger to be identified is a free radical scavengerfor use in cryopreserving mouse sperm, activity (a) and/or activity (c)are assessed. In one embodiment, if a free radical scavenger to beidentified is a free radical scavenger for use in cryopreserving bovinesperm, activity (a) and/or activity (b) are assessed. The method can becarried out using a variety of approaches to detecting the effect, ifany, of a candidate free radical scavenger, such as a method in which adye is used. In one embodiment, the method comprises incubating (a)sperm loaded with a cell-permeant indicator (dye) for reactive oxygenspecies that is nonfluorescent unless oxidation occurs within the spermand (b) a candidate free radical scavenger, thereby producing anincubated combination and assessing the level of reactive oxygen specieswithin sperm in the incubated combination. If the level of reactiveoxygen species in the sperm is reduced (e.g., relative to the level ofreactive oxygen species within the sperm in the absence of the candidatefree radical scavenger), the candidate free radical scavenger is a freeradical scavenger suitable for use in cryopreserving sperm. A freeradical scavenger identified by assessment of one or two of the threeactivities described above, can be further assessed for its usefulnessas a free radical scavenger by assessing the additional activities. Forexample, if capability (ability) to reduce reactive oxygen species insperm is assessed, the effect of the free radical scavenger on spermmotility, its effect of the in vitro fertilization; or both can beassessed, as appropriate, to provide further indication of thesuitability of the free radical scavenger for use in cryopreservation ofsperm.

EXAMPLES

The following examples describe use of the media and methods of thepresent invention. They are not intended to be limiting in any way.

Example 1 Cryopreservation of Mouse Sperm

Historic reports have detailed that live born recovery rates whencryopreserved C57BL16 sperm are used typically range from 0% to lessthan 10% (Glenister and Thornton, 2000). Nagakata reports afertilization rate of 26% (Nakagata and Takeshima, 1993). Applying verycomplex procedures like the two-step method to select for higher qualitysperm at thawing by (Bath et al 2003 improve the fertilization rates,but are not adequate for efficient banking (including recovery) of largenumbers of C57BL/6 strains via cryopreserved spec in.

The Jackson Laboratory has been working on developing a viable,economical approach to archive inbred and especially C57BL/6 strains.Described herein is a modified cryoprotective media (CPM) that providesan economical approach to this problem.

TABLE 2 Comparison of Cryopreservation Approaches: Characterization andFertilization Capacity of C57BL/6J Sperm. Post-thaw % Intact Post-thaw %Intact CPM Acrosomes Membranes % 2-cell Traditional 16.1 ± 0.06 22.5 ±0.08 7.03 ± 0.35^(b) method CPM 16.8 ± 0.13 26.4 ± 0.22 19.2 ± 0.26^(a)method

CByB6F1 females were used for oocyte donation. Comparisons of percentswere made using an arcsine transformation; percents within a columnhaving unique superscript (a and b) are different with Tukey-Kramer HSD(p<0.05); (see Table 2). Table 2 shows that no measurable differenceswere noted in mouse sperm viability or in the percentage of livespermatozoa having intact acrosomes, but that there is a significantdifference in the mouse sperm fertilization rate (% 2-cell) with 19%using the CPM versus 7% using the traditional method.

Applicants have shown that fertilization rates are greatly andconsistently improved when the media and methods described herein arecarried out. The epididymides and vas deferentia are extracted from theC57BL/6J male and placed into the cryoprotective media described herein.They are extracted and placed into an incubated 1 mL collection drop ofCryoProtective Medium (CPM), which comprises, for example, raffinose[18% w/v], skim milk [3% w/v] and monothioglycerol (MTG) atconcentrations from 159 μM to 636 μM. Incisions are made in the tissues,allowing the sperm to swim out into the CPM; this is allowed for atleast 5 but for less than 20 min. The residual tissues are then removedfrom the collection drop. Ten micro liters of the sperm sample areloaded into as many as one hundred 250 μL French straws (IMV; MapleGrove, Minn.). Two hundred and fifty microliter straws, containing a 5.5cm column of CPM, a 2.5 cm column of air, a 0.6 cm column of spermsample followed by a column of air, are sealed with an instantaneousheat sealer. The straws are loaded into cassettes and exposed to liquidnitrogen vapor for at least 10 min before being plunged into the liquidnitrogen. This essentially cools the sperm at 37° C./min.

Subsequent to storage in liquid nitrogen, 3 samples from each treatmentare thawed in a 37° C. water bath for 30 sec, and then each 10 μLaliquot is placed directly into its own 500 μL in vitro fertilization(IVF) drop of Cooks Mouse Vitro Fert (Cook Australia; Queensland,Australia). After an hour of incubation, 4 cumulus intact oocyteclutches from superovulated C57BL/6J females are added to the in vitrofertilization drops. IVF is carried out using methods known to those ofskill in the art, such as by the methods described by Nakagata.(Nakagata, 2000a; Nakagata, 2000b). The sperm and oocytes are incubatedtogether for 4 hrs before the presumptive zygotes are removed from theIVF drop and placed in a culture drop for overnight incubation.

Approximately 18 hours later, the percentage of oocytes developing into2-cell embryos is determined (#2-cell embryos/# total oocytes). Forstatistical analysis, percents are arcsine transformed and the averagesof at least 3 IVF drops per treatment are evaluated with a T-Test. Adifference between treatments is illustrated with an asterisk (p<0.05).For presentation, means and standard errors were converted back intopercents.

All concentrations of MTG show an improved fertilization rate, relativeto results obtained using presently-available methods. An MTGconcentration of 477 μM provided the best results. See FIG. 1 and Table3 and Table 4A.

TABLE 3 Fertilization Rate using C57BL/6J Mice and Either CPM orTraditional Method 2-Cell Embryos Fertilization Rate Transferred LiveBorn CPM 68.4% 225 80 Traditional method 6.5% 45 7

The following day the 2-cell embryos can be transferred topseudopregnant recipients or cryopreserved for later use.

Previous methodologies have provided fertilization rates of lower than10% (Songsasen and Leibo, 1997; Szczygiel et al., 2002; Sztein et al.,2001; Thornton et al., 1999) when using cryopreserved C57BL6J sperm instandard IVF procedure without any manipulation of the oocyte andwithout preselection of the sperm, not using intracytoplasmic sperminjection (ICSI). There is only one report which claims to have achieved26% fertilization rate for C57BL6/J (Nakagata and Takeshima, 1993).Employing the approach described herein, which includes the use of freeradical scavenger, fertilization rates of greater than 75% have beenobtained. These methods have also greatly and consistently improved thefertilization rates observed for other mouse strains as well. Forexample, using previous methods it was not uncommon to findfertilization rates for BALB/cJ, FVB/NJ, and 129P3/J to be less than10%, 30% and 5%, respectively.

Mice referred to herein as 129P3/J were referred to in the priorityapplications as 129P/J mice. This is only a change in nomenclature. Themethod described herein has resulted in observed fertilization rates of58%, 98% and 48%, respectively. See Tables 4A and 4B.

TABLE 4A Assessment of Fertility Rates in Different Mouse Strains Strain129X1/SvJ 129S1/SvlmJ BALB/cByJ BALB/cJ C3H/HeJ FVB/NJ C57BL/6JTreatment Mean Fertilization Rate CPM + MTG 51.2% 18.5% 69.1% 58.4%99.0% 98.6% 68.4% (81/158) (80/425) (231/177) (153/290)  (301/307)(203/209) (326/488)  Traditional 21.7%  0.0%  7.0% 10.3% 55.3% 27.4% 6.5% Method (93/428) (0/36)  (39/521) (144/1299) (150/327) (203/607)(227/1581)

TABLE 4B Assessment of Fertility Rates in Different Mouse StrainsTraditional Mouse Strain CPM (+MTG) Method 129P3/J 48.20% 12% (Songsasenand Leibo, 1997) A/J 92.67% no data CAST/EiJ  2.38%  0% DBA/2J 87.85%45% FVB/NJ  98.6% 27.4%  NOD/LtJ 75.21% no data SJL/J 46.56% no data

Example 2 Cryopreservation of Mouse Sperm with CPM Containing GSH

Applicants have shown that fertilization rates are greatly andconsistently improved when the media and methods described herein arecarried out. The epididymides and vas deferentia are extracted from theC57BL/6J male and placed into the cryoprotective media described herein.They are extracted and placed into an incubated 1 mL collection drop ofCryoProtective Medium (CPM), composed of raffinose [18% w/v], skim milk[3% w/v] and reduced glutathione [GSH; 500 μM to 50 mM]). Incisions aremade in the tissues, allowing the sperm to swim out into the CPM; thisis allowed for at least 5 but for less than 20 min. The residual tissuesare then removed from the collection drop. Ten micro liters of the spermsample are loaded into as many as one hundred 250 μL French straws (IMV;Maple Grove, Minn.). Two hundred and fifty microliter straws, containinga 5.5 cm column of CPM, a 2.5 cm column of air, a 0.6 cm column of spermsample, followed by a column of air, are sealed with an instantaneousheat sealer. This essentially cools the sperm at 37° C./min. After beingstored in liquid nitrogen, 3 samples from each treatment are thawed in a37° C. water bath for 30 sec, and then each 10 μL aliquot is placeddirectly into its own 500 μL in vitro fertilization (IVF) drop of CooksMouse Vitro Fert (Cook Australia; Queensland, Australia). After an hourof incubation, 4 cumulus intact oocyte clutches from superovulatedC57BL/6J females are added to the in vitro fertilization drops.(Nakagata, 2000a; Nakagata, 2000b).

The sperm and oocytes are co-incubated for 4 hrs before the presumptivezygotes are removed from the IVF drop and placed in a culture drop forovernight incubation.

Approximately 18 hours later, the fertilization rate is determined(#2-cell embryos/# total oocytes). For statistical analysis, percentsare arcsine transformed and the averages of the 3 IVF drops pertreatment are compared to the control of no GSH using Dunnett's methods.Results are presented in FIG. 2. A significant difference from thecontrol is illustrated with an asterisk. For presentation, means andstandard errors are converted back into percents.

Testing different concentrations of GSH reveals that the concentrationsfrom 500 μM to 5 mM increase the fertilization rates compared to CPMwithout a reducing agent. Use of 500 μM GSH shows an enhancement infertilization rates similar to those obtained with 477 μM MTG undersimilar conditions.

Example 3 Fertilization Rates with Cryopreserved Sperm from VariousMouse Strains Differ in Response to Cryopreservation with MTG.

Ten μL aliquots of sperm from a variety of mouse strains are frozen inthe absence (CPM-MTG) or presence of monothioglycerol (CPM). A total ofat least 6 sperm samples per treatment within a strain are utilized.These represented at least 3 samples from each of at least 2 sets of 2males whose sperm are pooled at the time of collection and then splitinto the two treatments. Subsequent to thawing, the samples are placeddirectly into their own 500 μL in vitro fertilization (IVF) drop ofCooks Mouse Vitro Fert (Cook Australia; Queensland, Australia; (Quinn etal., 1995)). After an hour incubation, 4 cumulus intact oocyte clutchesfrom isogenic strains are added to the incubation medium. Following a 4hr co-incubation, the presumptive zygotes are washed and culturedovernight. Approximately 18 hours later, the fertilization rate isdetermined (#2-cell embryos/# total oocytes). See Table 5.

TABLE 5 Fertilization Rates Determined in the Presence (CPM) and Absenceof MTG (CPM-MTG) Strain 129S1/SvlmJ BALB/cJ C57BL/6J TreatmentFertilization Rate CPM-MTG 31.1% 46.2% 49.0% CPM 48.1% 58.4% 68.4%

Example 4 Assessment of Reduction of Reactive Oxygen Species (ROS) byMTG Description of Free Radical Scavenger by Dye/Sperm Method

Sperm cryopreserved in the presence and absence of the free radicalscavenger, the reducing agent monothioglycerol (MTG), are loaded withthe green fluorescent dye CM-H2DCFDA to measure intracellular reactiveoxygen species. The sperm are challenged with varying levels ofTert-Butylhydrogen peroxide to induce oxidative damage and greenfluorescence is measured using flow cytometry. As shown in FIG. 3, MTGreduces the ROS in cryopreserved sperm from both C57BL/6J (B6) andBALB/cByJ (BALB/cBy) mice.

Example 5 Cryopreservation and Recovery of Sperm from GeneticallyModified Mouse Strains

The same approaches have been carried out on sperm obtained fromC57BL/6J males having genetic modifications and variable geneticcontributions from the 129 strain, as well as other strains, such asFVB/N.

Ten μL aliquots of sperm from a number of genetically modified mousestrains are frozen in CPM with 477 μM monothioglycerol as the freeradical scavenger (CPM) according to the method as described inExample 1. For a transgenic mouse carrying a YAC expression construct,sperm from the strain Tg(YAC72)2511Hay on the FVB/NJ background wascollected. For mice carrying modifications due to gene targeting(knockout) sperm from the mouse strains PLAU knockout, glycogen synthasekinase 3β knockout and solute carrier family 22 organic/anion cationtransporter member 12 knockout was collected. The strain backgroundmeans here a contribution of 50% or more of this strain to the geneticbackground of the mouse strain. The fertilization rate was determined(#2-cell embryos/# total oocytes) and the average percentage wascalculated. The values within the parentheses illustrate the total # oftwo cells/the total number of oocytes across IVFs. As shown in Table 6,high fertilization rates have been obtained in all cases. Thisdemonstrates that cryobanking male mouse germplasm for strainreconstitution is a viable option.

TABLE 6 Fertilization Capacity of Sperm from Genetically Modified Mice.JR % 2-Cell Stage Reference Strain (# 2-cell/# No. # Background GeneticModification oocytes) 3640 FVB/NJ Tg(YAC72)2511Hay  81 (105/130)transgene 3238 BALB/cJ C2ta (class II transactivator)  76 (112/148)knockout 2329 FVB/NJ PLAU (plasminogen activator, 86 (15/176) urokinase)knockout 5817 C57BL/6J Glycogen synthase kinase 3β 65 (72/111) knockout5839 C57BL/6J Solute carrier family 22 68 (53/78)  organic/anion cationtransporter member 12 knockout

Example 6 Determination of Sperm Concentration and Motility

To determine the concentration, motility, and progressive motility of“freshly-collected” sperm, the sperm is typically diluted 1:20 in eitherphosphate buffered saline or Cooks Mouse Vitro Fert (Cook Australia;Queensland, Australia; (Quinn et al., 1995)). To assess thawed sperm,sperm is incubated in IVF medium for an hour, at a dilution of 1:50 inthe IVF drop. It is then loaded into a Hamilton Thom IVOS computerizedsemen analyzer (Hamilton Thom, Beverly, Mass.). For measurement, thecalibration parameters are set as follows:

-   -   Apply Sort: 2    -   Frames acquired: 30    -   Frame rate: 60 Hz    -   Minimum contrast: 30    -   Minimum Cell Size: 4 pixels    -   Minimum Static Contrast: 15    -   Straightness (STR), Threshold: 50%    -   Low VAP Cutoff: 10.0 μm/s    -   Medium VAP Cutoff: 50.0 μmis    -   Low VSL Cutoff: 0.0 grids    -   Head Size, Non-Motile: 13 pixels    -   Head Intensity, Non-Motile: 75    -   Static Head Size: 0.57 to 2.91    -   Static Head Intensity: 0.14 to 1.84    -   Static Elongation: 0 to 87    -   Slow Cells Motile: YES    -   Magnification: 0.82    -   Video Source Camera    -   Video Frequency: 60    -   Bright Field No    -   Brightness for LED: 2663    -   Brightness for Ident: 3000    -   Temperature, Set: 37° C.

Apply Sort:  2 Frames acquired: 30 Frame rate: 60 Hz Minimum contrast:30 Minimum Cell Size: 4 pixels Minimum Static Contrast: 15 Straightness(STR), Threshold:   50% Low VAP Cutoff: 10.0 μm/s Medium VAP Cutoff:50.0 μm/s Low VSL Cutoff:  0.0 μm/s Head Size, Non-Motile: 13 pixelsHead Intensity, Non-Motile: 75 Static Head Size: 0.57 to 2.91 StaticHead Intensity: 0.14 to 1.84 Static Elongation:  0 to 87 Slow CellsMotile: YES Magnification:    0.82 Video Source: Camera Video Frequency:60 Bright Field: No Brightness for LED: 2663  Brightness for Ident:3000  Temperature, Set: 37° C. Cell Type: User Cell Depth Setup: 100.0μm Field Selection Mode: SELECT Indent Active: NO Ident Mode: BIntegrating Time: 1 Frames Optics Video Gain: Medium Video Brightness:2300  Video Contrast: 40 Video Sync Level: FW Sync_Level_100 VideoVertical Sync: 75 Sort #2 Points in Track:  16 to 100 Track Speed (VCL): 146.0 to 1000.0 Linearity (LIN):  0.0 to 20.0

Example 7 Assessment of Sperm Fertilization Capability AfterCryopreservation

To ensure that the enhancement in C57BL/6J sperm fertilizationcapability post cryopreservation is not just to the development of twocell embryos, Applicants also transferred embryos to pseudopregnantrecipients. Of the 225 embryos transferred, from a total of 3 pools of 3males, 80 developed to term (35.6%). This is approximately a three tofive fold enhancement over previous data obtained in Applicants' andmany other IVF labs with cryopreserved C57BL/6 sperm (FIG. 4).

Example 8 Cryopreservation of Sperm from Genetically Modified Mice withCPM Containing MTG and Recovery to Live Born

Ten μL aliquots of sperm from a variety of mouse strains as listed inTable 7 are frozen in the CPM with 477 μM monothioglycerol as ROS(CPM).A total of at least 6 sperm samples per treatment within a strain areutilized. These represented at least 3 samples from each of at least 2sets of 2 males whose sperm are pooled at the time of collection andthen split into the two treatments. Subsequent to thawing the samplesare placed directly into their own 500 μL in vitro fertilization (IVF)drop of Cooks Mouse Vitro Fert (Cook Australia; Queensland, Australia;(Quinn et al., 1995)). After an hour incubation, 4 cumulus intact oocyteclutches from isogenic strains were added to the incubation medium.Following a 4 hr co-incubation, the presumptive zygotes were washed andcultured overnight. Approximately 18 hours later, the fertilization ratewas determined (% 2 cell: #2-cell embryos/# total oocytes, see Table 7).The average percentage was determined. For each line, about 30 2-cellembryos were implanted into receptive females. After approximately 21days, the number of live born mice was determined, shown as % Live bornin Table 7.

In summary, the procedures described herein, in which cryoprotectivemedia as described above was used, give good and consistent results forgenetically modified mouse sperm, comparable to the parental inbredstrains. With the parental strains, the following fertilization rateshave been obtained: 129S1/SvImJ 18%; C57BL/6J 68%; Balb/cJ 58%, forBALB/cByJ 69%, and FVB/NJ 98%.

TABLE 7 # 2-Cell % 2-Cell % Live Sperm Donor Oocyte Donor # OocytesEmbryos Embryos born C57BL/6-Tg(Csf1r-EGFP- C57BL/6J 219 59 26.94 43.3NGFR/FKBP1A/TNFRSF6)2Bck/J B6(Cg)-Ncf1<m1J>/J C57BL/6J 218 84 38.53 43.3B6.Cg-Mapt<tm1(EGFP)Klt> Tg(MAPT)8cPdav/J C57BL/6J 224 54 24.11 30B6.129S-Shh<tm2(cre/ESR1)Cjt>/J C57BL/6J 182 142 78.02 26.7B6;C3H-Tg(Scgb1a1-Scnn1b)6608Bouc/J B6C3FeF1 209 180 86.12 66.7CByJ.A-Ttc7<fsn>/J BALB/cByJ 221 176 79.64 46.7B6.129S2(C)-Stat6<tm1Gru>/J C57BL/6J 223 223 100 43.3C3.B6-Tg(Fabp1-Ccnd1)4Rdb/J C3H/HeJ 138 107 77.54 26.7B6.129S1-Csf2rb1<tm1Cgb>/J C57BL/6J 253 37 14.62 35.7B6.HRS(BKS)-Cpe<fat>/J C57BL/6J 237 116 48.95 13.3 B6.D2N-Ahr<d>/JC57BL/6J 143 67 46.85 40 B6.Cg-F2rl1<tm1Mslb>/J C57BL/6J 186 95 51.08 50B6.129P2-P2rx7<tm1Gab>/J C57BL/6J 242 79 32.64 50FVB-Tg(Sod1-G86R)M1Jwg/J FVB/NJ 134 40 29.85 48 C.Cg-Fv4<r>/HmJ BALB/cJ138 21 15.22 28.6 C.Cg-Gata1<tm6Sho>/J BALB/cJ 70 61 87.14 20 STOCKDicer1<tm1Bdh>/J C57BL/6J 217 128 58.99 46.7 B6.Cg-Gusb<mps>/BrkJC57BL/6J 289 60 20.76 33.3 B6.129S4-Cxcl10<tm1Adl>/J C57BL/6J 257 6424.90 50 B6.Cg-Msr1<tm1Csk>/J C57BL/6J 161 35 21.74 53.3 B6;129S7-Ephb4<tm1And>/J C57BL/6J 285 100 35.09 36.7 B6.Cg-Tg(F2RL1)1Mslb/JC57BL/6J 235 113 48.09 33.3 B6.129S2-Thbs1<tm1Hyn>/J C57BL/6J 204 6230.39 56.7 B6.Cg-Tg(PDGFB-APPSwlnd)20Lms/2J C57BL/6J 232 55 23.71 36.7B6.C3-Gusb<mps-2J>/BrkJ C57BL/6J 188 57 30.32 30B6.C-H2<bm1>/ByBir-Gusb<mps>/SopJ B6.C-H2<bm1>/ByJ 94 22 23.40 47.6B6.Cg-Tg(ACTB-EGFP)1Osb/LeySopJ C57BL/6J 299 116 38.80 36.7B6.129S6-Ppt1tm1Hof/J C57BL/6J 227 121 53.30 30C3H/HeOuJ-Gusb<mps-2J>/BrkJ C3H/HeOuJ 211 134 63.51 36.7C57BL/6J-Kit<W-41J>/SopJ C57BL/6J 304 109 35.86 53.3129-Gt(ROSA)26Sor<tm1Luo>/J 129/SvlmJ 281 85 30.25 30CBy.Cg-Gpi1<a-m1Ehs>/BrkJ BALB/cJ 141 25 17.73 58.3B6.Cg-Gpi1<a-m1Ehs>/BrkJ C57BL/6J 195 49 25.13 20

Example 9 Cryopreservation of Rat Sperm

The epididymides and vas deferentia were collected from one 33 week oldLEW/SsNHsd rat (Harlan, Indianapolis, Ind.). Incisions were made in thetissue and the sperm were allowed to swim out, into 2 mL of a solutioncontaining 8% lactose [w/v] (Sigma Aldrich; St. Louis, Mo.) and 23% eggyolk [v/v] from fresh eggs in distilled water (Invitrogen; Carlsbad,Calif.) for 10 min. The sperm suspension was then split with half beingplaced into the lactose/egg yolk solution and the other half beingsuspended in lactose/egg yolk solution containing 954 micromol MTG in 15mL conical tubes. This resulted in a final MTG concentration of 477 μM.The 15 mL conical tubes containing the sperm samples were placed into abeaker containing ˜150 mL of 15° C. water for 10 min. After the initialcooling period, the beaker containing the water and conical tubes wastransferred to a 4° C. refrigerator where the samples were cooled for anhour and 15 min. After cooling to 4° C., the sperm was diluted into sixdifferent media compositions as described in Table 8. The CPM for ratsperm had the following composition: cryoprotectant was lactose alone orlactose and glycerol or propylene glycol; the membrane protectant wasfresh chicken egg yolk and MTG as the free radical scavenger.

The sperm were loaded into 250 μL French straws (IMV; Maple Grove,Minn.; cat# AAA201), which were then sealed with an instantaneous heatsealer (model AIE-305HD; American International Electric; Whittier,Calif.) and 5 of them loaded into cassettes (Zanders Medical Supplies;Vero Beach, Fla.). The cassettes were placed onto a raft situated withina styrofoam box containing liquid nitrogen and the samples were exposedto LN₂ vapor for at least 10 min before being plunged into the liquidphase. The apparati employed essentially allowed the sperm to be cooledat 37° C./min from a temperature of −10° C. to a temperature of −60° C.Sperm samples were stored in liquid nitrogen for at least 20 hours. Thesamples were thawed in a 37° C. water bath and pushed out the straw intoa 35×10 mm standard BD Falcon petri dish (Fisher Scientific, USA). Thesperm were diluted 1:50 in Cooks Mouse Vitro Fert (Cook's; Spencer,Ind.) and motility was evaluated by counting at least 5 different fieldsusing a video monitor connected to a microscope using a magnification of40×. The following values were used: 0 for no motility, + to ++++ forthe proportion of motile cells with ++++ being the highest. In all casestested, the addition of MTG yielded an increased motility with the CPMcomposed of lactose, glycerol, egg yolk and MTG giving the best result.

TABLE 8 Analysis of Rat Sperm Motility after Cryopreservation in aNumber of Different Media. CPM composition Membrane Free RadicalMotility Response Cryo Protectant Protectant Scavenger after Thawing 8%lactose 23% egg yolk + 8% lactose 23% egg yolk 477 μM MTG + 8% lactose,0.9M 23% egg yolk ++ glycerol 8% lactose, 0.9M 23% egg yolk 477 μM MTG++++ glycerol 8% lactose, 0.9M 23% egg yolk 0 propylene glycol 8%lactose, 0.9M 23% egg yolk 477 μM MTG +++ propylene glycol

Example 10 Cryopreservation of Bovine Sperm

Semen was collected via an artificial vagina from 6 different Holsteinbulls over 3 different days. The bulls were selected such that threeproduce sperm with good consistent freezing quality and three producesperm that respond to cryopreservation with poorer consistency.Antibiotics were added to the neat semen and initial volume andconcentration were determined using a graduated cylinder andspectrophotometry. From each collection, 5 different 500 μL aliquots ofsemen were extended with 1.5 mL of Triladyl egg yolk extender (Minitube;Verona, Wis.) containing glycerol. Subsequently, 2 mL of the sameextender containing no MTG, 200, 1000, 2000 or 10,000 μM MTG was addedto the extended semen to yield the final concentrations for MTG of 0,100, 500, 1000 or 5000 μM. After extension, the semen was transferred toa cold room and cooled to 4° C. over ˜1.5 hours. Extender containing theappropriate 1× concentration of MTG was supplied to each aliquotbringing the final sperm concentration to 80×10⁶/mL. This extended plussemen was loaded into ¼CC French Straws using automated fillers (MRS4;IMV technologies, Maple Grove, Minn.) and stored at 4° C. for 3 hrs.Following the equilibration time period, the straws were frozen in acontrolled rate freezer as follows: 4° C./min to −12° C.; 40° C./min to−100° C.; 20° C./min to −140° C. The samples were transferred to gobletsand stored in liquid nitrogen until examination.

For the examination, 6 straws per treatment per collection were thawedin a 37° C. water bath for at least 60 sec. For each treatment within acollection, two pools of semen were created and analyzed by combiningthe contents of three straws. For motility, the sperm samples werediluted 1:3 in phosphate buffered saline (PBS) and stained with 100μg/mL of the fluorescent DNA stain Hoechst 33342 before they wereexamined using the Hamilton Thorne Integrated Visual Optics System(IVOS). The percent of motile sperm was determined on 4 different fieldswithin each of 2 separate chambers. For viability the sperm samples werediluted 1:100 in PBS and stained with SYBR 14 (00 nM) and propidiumiodide (12 μM About 10,000 cells were counted using the Partec FlomaxFlow Cytometer (Ft. Collins, Colo.).

Results:

Motility. No differences were detected in motility among the treatments.This non-discrepancy was maintained even after 3 hr of incubation.Similarly, when bulls were grouped based on their sperm freezabilitycharacteristics, no differences at 0 or 3 hrs were detected. Theseresults show that the CPM consisting of glycerol, egg yolk and MTG atvarious concentrations has no ill effects on the overall post-thawmotility of bull sperm.

Viability. As shown in FIG. 5C, the overall viability was not negativelyinfluenced by the CPM consisting of glycerol, egg yolk and MTG atvarious concentrations. When individual bulls were analyzed individually(FIG. 5C) positive effects appear evident.

Addition of MTG to the cryopreservative media results in increase ofviability with a slight effect for the 3 bulls and about an 8% increasefor the other 3 bulls.

Example 11 Human Semen Cryopreservation

One semen sample is collected from each of healthy, normal, fertilemales. Fertile men are defined as individuals aged 19-30 who havenaturally fathered a child within the past 6 months. Furthermore, thesamples are considered normal if they meet the parameters set by WHOcriteria for sperm count, motility, and morphology. The semen sample iscollected after a minimum of 48-72 hours of sexual abstinence, but notlonger than seven days. The semen is obtained by masturbation andejaculated into a sterile, wide-mouthed plastic container that has beentoxicity tested. The subject is instructed that care must be taken toinclude the entire specimen in the container. Upon receipt of theejaculate by the laboratory, each semen sample is entered in a semenanalysis logbook. The semen sample is assigned a consecutive accessioncode number, which is recorded along with the patient's name and thedate of collection. The patient's sample is identified by the accessioncode number throughout the freezing procedure; all tubes, slides, etc.are labeled with this number. While the sample liquefies, thecryoprotective media is freshly prepared: TYB (Irvine Scientific, cat.#90128: 20% egg yolk—from USDA certified SPF (Virus Free) laying flocks,heat inactivated at 56° C. for 30 minutes; 12% v/v Glycerol; 10 μg/mLGentamicin Sulfate) and 477 μM MTG or TYB alone. For control TYB mediawithout MTG is used.

The semen sample is gently mixed with the freshly preparedcryoprotective media at a 1:1 ratio and 0.25 mL volumes of thesample/cryoprotectant mixture is transferred into each of the labeledcryovial (1 ml from Nunc). The cryovials are placed into a metal rackand is suspended in the LN₂ vapors of the storage tank for 12 minutes.After 12 minutes, the cryovials are placed onto the labeled, chilledaluminum cane, and the cane is inserted into the labeled, chilledcardboard sleeve and plunged into a canister in the storage tank. Thecryopreserved semen will be stored for at least 1 hour in the liquidnitrogen. A single vial will be thawed by placing the cryovial into abeaker containing 37° C. warm water for 10 minutes. Then the sample isgently mixed using a sterile pipette. The semen is analyzed formorphology (e.g. acrosome), motility, progression, viability, and countimmediately after thawing. The zona free hamster oocyte penetration testor the chlorotetracycline capacitation assay can be performed todetermine the quality of the semen.

Example 12 Preparation and Storage of Cryoprotective Media

Approximately 80 mL of bottled distilled water (Invitrogen, cat#15230-238) was placed in a beaker and heated for 40 sec in a microwaveon high power. Care was taken not to boil. The beaker was placed on aheated stir plate and 18 g of raffinose (Sigma; cat #R7630) was added.The solution was heated and stirred until it cleared. Subsequently, 3 gof skim milk (BD Diagnostics; cat #232100) was added and the solutionwas stirred until the skim milk powder dissolved. The solution wastransferred to a volumetric flask and brought to 100 mL with steriledistilled water (Invitrogen, cat #15230-238). Again, the solution wasmixed well and divided into two 50 mL centrifuge tubes. To clarify thesolution, it was centrifuged at 13,000×g for 15 minutes at roomtemperature (22° C.) and then filtered through a 0.22 μm cellulosefilter. This resulted in an 18% raffinose/3% skim milk solution having afinal osmolarity of 488 mOsm. One third of this solution wassupplemented with 477 μM monothioglycerol (MTG; Sigma cat #M6145) andfrozen at −80° C. in an Elite®. Series Revco Freezer (Asheville, N.C.).The other two thirds were frozen at −80° C. without any supplementation.

After 8 weeks of storage at −80° C., the raffinose skim milk solutionswere thawed in a 37° C. water bath until no more precipitate wasobserved (˜1 hr). The first cryoprotective media consisted of 18%raffinose, 3% skim milk, 477 μM MTG frozen for 8 weeks (FROZEN MTG). Thesecond cryoprotective media consisted of 18% raffinose and 3% skim milkfrozen for 8 weeks being supplemented at the time of sperm collectionwith 477 μM fresh MTG (FRESH MTG). The third cryoprotective mediaconsisted of 18% raffinose and 3% skim milk which had been frozen at−80° C. for 8 weeks (NO MTG).

Eighteen C57BL/6J males were sacrificed and their vas deferentia andepididymides were obtained and sperm was isolated as previouslydescribed (see Example 1). Nine different groups, of two males each,were created and each group was assigned to have their sperm collectedinto one of three different cryoprotective media. This resulted in 3groups of males being collected for each treatment. Four aliquots, of 10μL each, were loaded into ten ¼ CC French straws (IMV; Maple Grove,Minn.; cat# AAA201). These were then sealed with an instantaneous heatsealer (model ATE-305HD; American International Electric; Whittier,Calif.) and 5 of them loaded into a cassette (Zanders Medical Supplies;Vero Beach, Fla.). The cassettes were placed onto a raft situated withina Styrofoam box containing liquid nitrogen and the samples were exposedto LN₂ vapor for at least 10 min before being plunged into the liquidphase. This allowed the sperm to be cooled at 37° C./min from atemperature of −10° C. to a temperature of −60° C. The sperm sampleswere stored in liquid nitrogen for approximately 3 months.

To determine if storage of the cryoprotective media at −80° C. alteredthe activity of the C57BL/6J mouse sperm., in vitro fertilizations (IVF)were preformed as described previously (see Example 1, also seepublications by Nakagata, 2000a; Nakagata, 2000b, Sztein et al. 2000;Byers et al. 2006). Briefly the sperm and oocytes, both from C57BL/6Jmice, were incubated together for 4 hrs in a benchtop incubator at 37°C. After 4 hrs of co-incubation the presumptive zygotes were washedthrough two 150 μL drops of IVF media (MVF; Cook's; Spencer, Ind.) andthen cultured overnight. Approximately 18 hrs later, the proportion ofoocytes fertilized was calculated by dividing the number of two-cellembryos by the sum of two-cells and normally appearing presumptiveone-cell oocytes.

FIG. 6 clearly demonstrates that storing the cryoprotective media withMTG for 2 months at −80° C. performs equally compared to cryoprotectivemedia with freshly added MTG. The column FROZEN MTG shows thefertilization rate when C57BL/6J sperm was cryopreserved with thecomplete media being stored at −80° C. The column FRESH MTG shows thefertilization rate when C57BL/6J sperm was cryopreserved with mediabeing stored at −80° C., but MTG being freshly added. The column NO MTGshows the fertilization rate when C57BL/6J sperm was cryopreserved withmedia being stored at −80° C., but not containing MTG freshly.Differences in the percent of oocytes developing to 2-cell among thetreatments were determined using analysis of variance on arcsinetransformed percents and comparisons for all pairs of means usingTukey-Kramer HSD (JMP; SAS Institute, Cary, N.C.) statistical analysis.Those means with different letters are significantly different (p<0.05).For presentation, the average and standard deviations of the percentsare shown. No significant difference between the two media preparationsFROZEN MTG and FRESH MTG can be observed.

Example 13 Identification of a Suitable Free Radical Scavenger

A suitable free radical scavenger (useful for cryopreservation of sperm)can be identified by testing one or more of the following activities (a)capability to reduce Reactive Oxygen Species (ROS), (b) effect on spermmotility, and (c) effect on in vitro fertilization (IVF). In thoseinstances in which a suitable free radical scavenger for use with mousesperm is being identified, (a) and (c) are assessed/carried out. Forbovine sperm, (a) and (b) are carried out. In each instance, the thirdactivity ((b) for mouse and (c) for bovine) can be carried out. Inspecific embodiments, one of either (b) or (c) above will be tested, inaddition to (a) above.

For example, the capability to reduce ROS can be tested by a dye/spermmethod as described in Example 4 and also see publications by Nelid etal 2006 and Guthrie and Welch 2006. Either fresh or cryopreserved spermcan be used in this assay. For example, sperm collected from the vasdeferens and epididymis of two C57BL/6J mice into 1 mL of a solutioncontaining 3% skim milk (w/v; BD Diagnostics; cat #232100) and 18%raffinose (w/v; Sigma; cat #R7630). A fraction of the collected spermwas diluted 1:1 in the same skim milk raffinose solution while anotherfraction was diluted 1:1 using the skim milk raffinose solutionsupplemented with 954 mM monothioglycerol (MTG; Sigma Aldrich; cat#M6145). Another set of C57BL/6J males was used to collect sperm anddilute in the skim milk raffinose solution with 1 mM glutathione (GSH;Sigma Aldrich; cat #G6013) or 2 mM dithiothreitol (DTT; Sigma Aldrich;cat #646563). The sperm was cryopreserved as described in Example 1 andstored in liquid nitrogen for at least 24 hours.

To determine if MTG, GSH or DTT can act as a free radical scavenger, thesperm were thawed and loaded with the green fluorescent dye5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate(CM-H2DCFDA; Invitrogen C6827). This dye is a cell-permeant indicatorfor reactive oxygen species that is nonfluorescent until removal of theacetate groups by intracellular esterases and oxidation occurs withinthe cell. The emission of fluorescence correlates with ROS generation asa result of DCFH oxidation. To load the sperm, at least 150 μL of spermwere thawed and diluted 1:1 in a sperm incubation media described byBath et al. ((Bath 2003); 123 mM NaCl; 4.7 mM KCl; 0.2 mM MgSO_(4.7)H₂O;10 mM;NaHCO₃ 21 mM HEPES; 0.33 mM sodium pyruvate; 4.6 mM glucose; 100IU/mL penicillin G; 1.33 mg/mL BSA). The lyophilized dye was resuspendedin dimethylsulfoxide to a concentration of 432.5 μM and then added tothe cells, such that the final dye concentration was 40 μM. The spermwere allowed to take up the stain for 30 min and then 50 μL of the cellswas aliquoted into Fluorescent-activated cell sorting (FACS) tubes(12×75 mm tubes polystyrene, Falcon #2052 or 2054 or polypropylenetubes, USA Plastics PN 1450-0000). Five hundred microliters of spermincubation media containing a 50 fold dilution of the potential reducingagent used to cryopreserve sperm was added. This level of reducing agentwas selected to reflect the same 50 fold dilution observed during aroutine in vitro fertilization. Then the sperm were centrifuged for 5min at 300×g, the supernatant was decanted and the cells wereresuspended in 100 μL of sperm incubation media containing the 50 folddilution of the appropriate potential reducing agent. The sperm werethen challenged with various concentrations of tert-Butyl hydroperoxide(e.g. Sigma Aldrich; cat #B2633) to induce oxidative stress. Thestressed samples were incubated for 1 hour, brought to 300 μL in spermincubation media supplemented with the reducing agent being investigatedand then stained with 5 μg/mL of propidium iodide (e.g. Invitrogen, cat#P-3566) and 5 μg/mL of LDS-751 (Invitrogen, cat #L7595) todifferentiate nucleated live and dead cells. The samples were loadedonto either a FACScan (BD Biosciences; Franklin Lakes, N.J.) orFACSCalibur (BD Biosciences; Franklin Lakes, N.J.) flow cytometer andthe green fluorescence was measured on at about 5,000 viable sperm.FIGS. 7A and 7B show that all of the additives tested in this experimentreduced the level of reactive oxygen species within cryopreserved mousesperm cells.

FIGS. 7A and 7B show results of the following: C57BL/6 spermcryopreserved in the presence and absence of three potential freeradical scavengers (monothioglycerol (MTG); dithiothreitol (DTT);glutathione (GSH)) were loaded with the green fluorescent dye CM-H2DCFDAto measure intracellular reactive oxygen species (ROS). The sperm werechallenged with varying levels of tert-butyl hydroperoxide to induceoxidative stress and green fluorescence was measured using flowcytometry (A) FACSCalibur and (B) FACScan. As shown, the potentialreducing agents limited the level of ROS within the cryopreserved mousesperm. The y-axis shows the relative fluorescence being the highest whenno free radical scavenger is present (solid line) and the x-axis showsthe concentration of tert-butyl hydroperoxide.

If the compound is capable of reducing ROS at the tested concentration,then its effect on sperm motility and fertilization is tested. While thesperm motility assay is a helpful fast screening tool in the case ofmouse, this assay is not required for mouse and one can perform only theIVF assay in the case of mouse. For other species, e.g. bovine, thesperm motility assay together with the fluorescence assay is sufficient.The sperm motility assay can be done either with fresh or cryopreservedsperm. For example, the motility can be determined as described inExample 6 (Determination of Sperm Concentration and Motility), using anIVOS instrument. Briefly, C57BL/6 sperm were diluted 1:50 in pre-warmedand gassed mouse vitro fert (Cook's; Spencer, Ind.) under light mineraloil. The samples were incubated for an hour at 37° C., in a humidifiedatmosphere of 5% CO₂, 5% O₂ and 90% nitrogen in a K-MINC-1000 bench topincubator (Cook's; Spencer, Ind.). After incubation, the sperm weredrawn into 0.1×2.0 mm I.D. tubes (VitroCom Inc. Mt Lks. N.J.) usingcapillary action and 3 manually selected fields per sample were analyzedfor motility using the Hamilton Thorn (Beverly, Mass.) integrated visualoptics system (IVOS) for computerized semen analysis. Applicants haveshown that sperm frozen in CPM containing no free radical scavenger,0.477 mM MTG, 0.1 mM DTT, 1 mM DTT, 5 mM DTT, 20 mM DTT, 0.5 mM GSH, 1mM GSH, 5 mM GSH or 50 mM GSH can be assessed for motility and thatmotility of at least 5% is required to achieve fertilization in IVF. Allmeasurements were done in triplicate and the average is listed in Table9.

TABLE 9 C57BL/6 Sperm Motility after Different CPM Treatments AveragePost Thaw Free Radical Scavenger Motility IVF Fit none 10-29%  yes 0.1mM DTT 24.6% yes 1 mM DTT  34% yes 5 mM DTT 17.6% yes 20 mM DTT  1.6% no0.5 mM GSH  32% yes 1 mM GSH 25.3% yes 5 mM GSH 41.67%  yes 50 mM GSH 0.6% no 0.477 mM MTG  16% yes

If the compound has passed the sperm/dye test and the motility test,then its effect on IVF is tested. IVF was performed, as described inExample 1, with C57BL/6 sperm cryopreserved in the media containing asfree radical scavenger, DTT, GSH or MTG.

TABLE 10 57BL/6 Sperm Fertility After Different CPM TreatmentsFertilization Rate (2-cell Free Radical Scavenger embryos of totaloocytes) none 31.2% (56 of 179) 0.1 mM DTT 19.2% (33 of 169) 1 mM DTT 2%(2 of 94) 5 mM DTT 0.9% (2 of 212) 20 mM DTT 6.4% (11 of 147) 0.5 mM GSH59.7% (138 of 229) 1 mM GSH 49.5% (99 of 202) 5 mM GSH 39.9% (74 of 195)50 mM GSH 0.0% (0 of 249) 0.477 mM MTG 75.2% (111 of 149)

These data clearly show that sperm with a motility below 5% arecompromised and have less ability to fertilize oocytes (20 mM DTT and 50mM GSH). Further, these data show that, in addition to the motile spermcriteria, the fertilization rate results in a reliable assay todetermine the effectiveness of compound and its dose.

Example 14 Cryopreservation of Mouse Spermatozoa with CPM Containing aCryoprotectant and a Free Radical Scavenger (CP-FRS)

The epididymides and vas deferentia are extracted from three C57BL/6Jmales and placed into the cryoprotective media described herein. Spermare extracted and placed into an incubated 1 mL collection drop ofCryoProtective Medium (CPM), which comprises, for example, raffinose[18% w/v] and monothioglycerol [MTG: 100 μM to 1 mM] or raffinose [18%w/v] and reduced glutathione [GSH; 500 μM to 50 mM]). In this example,Applicants used 18% w/v raffinose and 477 μM monothioglycerol. The spermwere allowed to swim out into the CPM; this was allowed for at least 5minutes, but for less than 20 min. The residual tissues were thenremoved from the collection drop. Ten microliters of the sperm samplewere loaded into as many as one hundred 250 μL French straws (IMV; MapleGrove, Minn.). Four 10 μL aliquots of the collected sperm were loadedinto the straws, which were sealed with an instantaneous heat sealer.The straws were placed into cassettes and exposed to liquid nitrogenvapor for 10 minutes. This essentially cooled the sperm at 37° C./min.After being stored in liquid nitrogen for at least 24 hours, 6 samplesfrom each treatment were thawed in a 37° C. water bath for 30 sec, andthen each 10 μL aliquot was placed directly into its own 500 μL in vitrofertilization (IVF) drop of Cooks Mouse Vitro Fert (Cook Australia;Queensland, Australia: Quinn et al., 1995). After an hour of incubation,4 cumulus intact oocyte clutches from superovulated C57BL/6J femaleswere added to the in vitro fertilization drop (see Example 1 and alsoNakagata, 2000a; Nakagata, 2000b, Sztein et al. 2000; Byers et al.2006).

Briefly, the sperm and oocytes, both from C57BL/6J mice, were incubatedtogether for 4 hrs in a benchtop incubator at 37° C. After 4 hrs ofco-incubation the presumptive zygotes were washed through two 150 μLdrops of IVF media (MVF; Cook's; Spencer, Ind.) and then culturedovernight. Approximately 18 hrs later, the proportion of oocytesfertilized was calculated by dividing the number of two-cell embryos bythe sum of two-cells and normally appearing presumptive one-celloocytes.

TABLE 1 Animals for Which the Present Invention Can be Used CategoryExamples but not limited to: Notes: INBRED MOUSE LINES C3H CBA DBA FVBNOD BALB/c 129 C57BL C57BL Mice C57BL/6J, C57BL/6NTac, C57BL/6NCrl,C57BL/10 All other inbred mouse strains, 129S1/SvlmJ, 129T2/SvEmsJ,Definition: Inbred Mice are genetically including recombinant inbred129X1/SvJ, 129P3/J, A/J, AKR/J, homogeneous and homozygous at all loci.The strains BALB/cBy, BALB/cByJ, BALB/c International Committee onStandardized BALB/cJ, C3H/HeJ, C3H/HeOuJ, Nomenclature for Mice hasruled that a strain of C3HeB/FeJ, C57BL/10J, C58, mice can be considered“inbred” at generation CBA/CaHN-Btkxid/J, CBA/J, DBA/1J, F₂₀ DBA/1LacJ,DBA/2J, FVB/NJ, MRL/MpJ, NOD/LtJ, SJL/J, SWR/J, NOR/LtJ, NZB/BINJ,NZW/LacJ, RBF/DnJ, 129S6/SvEvTac, AJTAC, BALB/cAnNTac, BALB/cJBomTac,BALB/cABomTac, C57BL/6NTac, C57BL/6JBomTac, C57BL/10SgAiTac, C3H/HeNTac,CBA/JBomTac, DBA/1JBomTac, DBA/2NTac, DBA/2JBomTac, FVB/NTac,NOD/MrkTac, NZM/AegTac, SJL/JcrNTac, BALB/cAnNCrlBR, C3H/HeNCrlBR,C57BL/6NCrlBR, DBA/2NCrlBR, FVB/NCrlBR, C.B- 17/lcrCrlBR,129/SvPaslcoCrlBR, SJL/JorllcoCrlBR, A/JolaHsd, BALB/cAnNHsd,C3H/HeNHsd, C57BL/10ScNHsd, C57BL/6NHsd, CBA/JCrHsd, DBA/2NHsd,FVB/NHsd, SAMP1/KaHsd, SAMP6/TaHsd, SAMP8/TaHsd, SAMP10/TaHsd,SJL/JCrHsd, AKR/OlaHsd, BiozziABH/RijHsd, C57BL/6JOlaHsd, FVB/NhanHsd,MRL/MpOlaHsd, NZB/OlaHsd, NZW/OlaHsd, SWR/OlaHsd, 129P2/OlaHsd, and129S2/SvHsd. B6.129P2-Apoetm1Unc/J NOD.CB17-Prkdcscid/J 129S1/SvlmJ129X1/SvJ B10.A-H2a H2-T18a/SgSnJ B10.D2-Hc0 H2d H2-T18c/oSnJ B10.D2-Hc1H2d H2-T18c/nSnJ B10.RIII-H2r H2-T18b/(71NS)SnJ B6(C)-H2-Ab1bm12/KhEgJB6.129P2-ll10tm1Cgn/J B6.129P2-Nos2tm1Lau/J B6.129P2-Nos3tm1Unc/JB6.129S2-Cd8atm1Mak/J B6.129S2-Igh-6tm1Cgn/J B6.129S7-Ifngtm1Ts/JB6.129S7-Rag1tm1Mom/J B6.CB17-Prkdcscid/SzJ B6.MRL-Faslpr/J B6.V-Lepob/JBKS.Cg-m +/+ Leprdb/J C3HeB/FeJ C57BL/10J C57BL/10SnJC57BL/6-Tg(APOA1)1Rub/J C57BL/6J-Tyrc-2J/J CBA/CaHN-Btkxid/J CBA/CaJCBySmn.CB17-Prkdcscid/J FVB/N-Tg(MMTVneu)202Mul/J KK.Cg-Ay/J MRL/MpJMRL/MpJ-Faslpr/J SJL/J SWR/J B10.PL-H2u H2-T18a/(73NS)SnJ NONcNZO5/LtJWR BPH/2 BPL/1 FS P/J P/A PRO All genetically engineered (e.g. Examples:B6.129P2-Apoetm1Unc/J, transgenic, knockout, knockin,B6.129S4-Pdyn^(tm1Ute)/J, B6; 129P2- knockdown, retroviral, viral),Pemt^(tm1J)/J, NOD.Cg Prkdc^(scid)- chemically induced mutationsB2m^(b)/Dvs, (e.g. ENU, EMS also including archives of mutants);radiation induced mutations; spontaneous mutations/modificationsmaintained on mouse strains for example: C57BL/6, 129, FVB, C3H, NOD,DBA/2, BALB/c, CD-1. Including congenic strains and recombinant congenicstrains. Mouse hybrid strains produced Mice: by crossing two inbredstrains NZBWF1/J including mixed inbred strains B6CBAF1/J B6SJLF1/JCB6F1/J CByB6F1/J PLSJLF1/J WBB6F1/J-KitW/KitW-v B6129PF1/J CAF1/JB6129PF2/J B6129SF1/J B6129SF2/J B6AF1/J B6C3F1/J B6CBAF1/J B6SJLF1/J(including any other combination) Proportions vary from 50:50 F1 to 99:1Outbred Mice CD-1; ICR; Black Swiss; Swiss Definition outbred: Animalsfrom a defined Webster; NIH Swiss; CF-1, Nude population that areproduced by breeding schemes that avoid crosses between closely relatedindividuals in order to maintain the maximal level of heterozygosity inall offspring INBRED RAT LINES ACI, Brown Norway (BN), BCIX, Copenhagen(COP), MWF, D Agouti (DA), Goto-Kakizaki (GK), Lewis, Fischer 344(F344), Wistar Furth (WF), Wistar Kyoto (WKY; WKYN1), ZDF Allgenetically engineered (e.g. F344/NTac-Tg(HLA-B27)-Tg(2M)33- transgenic,knockout, knockin, 3Trg; HsdAmc: TR-Abcc2; knockdown, retroviral,viral), HsdHlr: ZUCKER-Leprfa; NIH Nude chemically induced mutations(e.g. ENU, also including archives of mutants); radiation- inducedmutations; spontaneous mutations/modifications maintained on rat strainsincluding congenic strains. Rat Hybrids Produced by BHR crossing twoinbred strains FBNF1/Hsd LBNF1/Hsd Outbred Rats Holtzman, SpragueDawley, Long Definition outbred: Animals from a defined Evans, Wistar,Wistar Han, WH, WKY, population that are produced by breeding Zucker,JCR (Russel Rat), OFA schemes that avoid crosses between closely relatedindividuals in order to maintain the maximal level of heterozygosity inall offspring All genetically engineered (e.g. Rabbit, goat, sheep, pig,cow, horse, transgenic, knockout, knockin, dog, cat knockdown,retroviral, viral), chemically induced mutations (e.g. ENU, alsoincluding archives of mutants); radiation induced mutations; spontaneousmutations/modifications maintained on mammals Other rodents Gerbils,Guinea pigs, Hamsters, Cotton Rat All genetically engineered (e.g. Fish(e.g. zebrafish, medaka, salmon, transgenic, knockout, knockin, tilapia,catfish, tuna); chicken, turkey knockdown, retroviral, viral),chemically induced mutations (e.g. ENU, also including archives ofmutants); radiation induced mutations; spontaneousmutations/modifications maintained on vertebrates Cows as outbred orrandom bred populations Pigs as outbred or random bred populations Goatas outbred or random bred populations Sheep as outbred or random bredpopulations Horses as outbred or random bred populations Humans asoutbred or random bred populations All other mammals Dogs, Cats,Camelids, Non-Human Primates, all outbred or random bred populations,zoo animals, marsupials, endangered mammals Other vertebrates Freshwaterfish (e.g. cyprinids, siluroids, salmonids, tilapia, medaka, zebrafish)Marine fish (e.g. herring, eel, sea bass, flounder, grouper, grunt,seatrout, drums, croaker, weakfish, dolphin, mackerels, tunas, snappers,swordfish, grouper, bluefish, bonefish, billfishes, tarpons, porgy,mullets, sharks, tarpons, tropical fishes); Endangered fish Birds(rooster, turkey, duck, goose, quail, pheasant, guinea fowl, pigeon,companion birds, endangered birds

REFERENCES

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The invention claimed is:
 1. A method for cryogenically preservingmammalian sperm, comprising adding to the mammalian sperm, prior tocryopreservation, a composition that comprises at least onecryoprotectant, at least one membrane protectant, and monothioglycerol,whereby mammalian sperm are effectively cryopreserved.
 2. The method ofclaim 1, wherein the cryoprotectant is selected from the groupconsisting of raffinose, lactose, trehalose, melibiose, melezitose,mannotriose, stachyose, dextran, sucrose, and sugar alcohols thereof,glycerol, maltitol, and lactitol.
 3. The method of claim 1, wherein thecryoprotectant is raffinose, lactose or glycerol.
 4. The method of claim1, wherein the membrane protectant is a protein selected from the groupconsisting of egg protein, egg yolk protein, egg white protein, casein,albumin, keratin, collagen, atelocollagen, elastin, gelatin, peptones,fibrinogen, fibronectin, a soy protein, a wheat protein, a corn protein,a milk protein, and hydrolysates thereof.
 5. The method of claim 1,wherein the membrane protectant is skim milk or a component thereof;milk powder or a component thereof; or egg yolk or a component thereof.6. The method of claim 1, wherein the mammalian sperm is isolated from amammal selected from the group consisting of: a mouse, a rat, a hamster,a guinea pig, a cat, a dog, a goat, a horse, a bull, a camel, a pig, arabbit, a sheep, and a human.
 7. The method of claim 1, wherein themammalian sperm is isolated from a rodent.
 8. The method of claim 1,wherein the mammalian sperm is isolated from a mouse.
 9. The method ofclaim 8, wherein the mouse is an inbred mouse strain or subs rain. 10.The method of claim 1, wherein the mammalian sperm is isolated from abovine.
 11. The method of claim 1, wherein the cryoprotectant israffinose, lactose, or glycerol; and wherein the membrane protectant ismilk protein, a protein in milk, egg protein, egg yolk protein or eggwhite protein.
 12. The method of claim 1, wherein the at least onecryoprotectant comprises at least one sugar or one sugar alcohol. 13.The method of claim 12, wherein the cryoprotectant is raffinose, lactoseor glycerol; and wherein the membrane protectant is egg yolk, skim milk,milk or a component thereof.
 14. The method of claim 1, wherein thecryoprotectant is selected from the group consisting of: a sugar, asugar alcohol and a combination thereof; and wherein the membraneprotectant is selected from the group consisting of: milk, milk powder,egg yolk and a combination of any two or more thereof.
 15. The method ofclaim 1, wherein the cryoprotectant is selected from the groupconsisting of: raffinose, lactose, glycerol and a combination of any twoor more thereof; and wherein the membrane protectant is selected fromthe group consisting of: milk, milk powder, egg yolk and a combinationof any two or more thereof.
 16. The method of claim 1, wherein thecomposition further comprises reduced glutathione, beta-mercaptoethanolor a combination thereof.
 17. A method for cryogenically preservingmammalian sperm, comprising adding to the mammalian sperm, prior tocryopreservation, a composition that comprises at least onecryoprotectant, at least one membrane protectant, and monothioglycerol,whereby cryopreserving the mammalian sperm in the composition providescryopreserved sperm active to produce a greater number of live bornoffspring compared to mammalian sperm of the same type cryopreserved inthe composition without monothioglycerol.
 18. The method of claim 17,wherein the cryoprotectant is selected from the group consisting ofraffinose, lactose, trehalose, melibiose, melezitose, mannotriose,stachyose, dextran, sucrose, and sugar alcohols thereof, glycerol,maltitol, and lactitol.
 19. The method of claim 17, wherein thecryoprotectant is raffinose, lactose or glycerol.
 20. The method ofclaim 17, wherein the membrane protectant is a protein selected from thegroup consisting of egg protein, egg yolk protein, egg white protein,casein, albumin, keratin, collagen, atelocollagen, elastin, gelatin,peptones, fibrinogen, fibronectin, a soy protein, a wheat protein, acorn protein, a milk protein, and hydrolysates thereof.
 21. The methodof claim 17, wherein the membrane protectant is skim milk or a componentthereof; milk powder or a component thereof; or egg yolk or a componentthereof.
 22. The method of claim 17, wherein the mammalian sperm isisolated from a mammal selected from the group consisting of: a mouse, arat, a hamster, a guinea pig, a cat, a dog, a goat, a horse, a bull, acamel, a pig, a rabbit, a sheep, and a human.
 23. The method of claim17, wherein the mammalian sperm is isolated from a rodent.
 24. Themethod of claim 17, wherein the mammalian sperm is isolated from amouse.
 25. The method of claim 24, wherein the mouse is an inbred mousestrain or substrain.
 26. The method of claim 17, wherein the mammaliansperm is isolated from a bovine.
 27. The method of claim 17, wherein thecryoprotectant is raffinose, lactose, or glycerol; and wherein themembrane protectant is milk protein, a protein in milk, egg protein, eggyolk protein or egg white protein.
 28. The method of claim 17, whereinthe at least one cryoprotectant comprises at least one sugar or onesugar alcohol.
 29. The method of claim 28, wherein the cryoprotectant israffinose, lactose or glycerol; and wherein the membrane protectant isegg yolk, skim milk, milk or a component thereof.
 30. The method ofclaim 17, wherein the cryoprotectant is selected from the groupconsisting of: a sugar, a sugar alcohol and a combination thereof; andwherein the membrane protectant is selected from the group consistingof: milk, milk powder, egg yolk and a combination of any two or morethereof.
 31. The method of claim 17, wherein the cryoprotectant isselected from the group consisting of: raffinose, lactose, glycerol anda combination of any two or more thereof; and wherein the membraneprotectant is selected from the group consisting of: milk, milk powder,egg yolk and a combination of any two or more thereof.
 32. The method ofclaim 17, wherein the composition further comprises reduced glutathione,beta-mercaptoethanol or a combination thereof.